Method for precipitating mono and multiple layers of organophosphoric and organophosphonic acids and the salts thereof in addition to use thereof

ABSTRACT

The invention relates to a method for precipitating mono or multiple layers of organophosphoric acids of the general formula I (A)
 
Y—B—OPO 3 H 2   (IA)
 
or of organophosphonic acids of the general formula I (B)
 
Y—B—PO 3 H 2   (IB)
 
and the salts thereof, wherein B is an alkyl, alkenyl, alkinyl, aryl, aralkyl, hetaryl or hetarylalkyl residue and Y is hydrogen or a functional group from the hydroxy, carboxy, amino, optionally low-alkyl-substituted mono or dialkylamino series, thiol, or a negative acid group from the ester, phosphate, phosphonate, sulfate, sulfonate, maleimide, succinimydyl, epoxy, acrylate series, wherein a biological, biochemical or synthetic indicator element may be coupled to B or Y by addition or substitution reaction, wherein compounds may also be added conferring the substrate surface a resistance to protein adsorption and/or to cell adhesion and in the B chain may optionally be comprised one or more ethylene oxide groups, rather than one or more —CH2— groups. According to the invention, said precipitation occurs on substrate surfaces of pure or mixed oxides, nitrides or carbides of metals and semiconductors, comprising the use of water-soluble salts of a compound of formula (IA) or (IB) for the treatment of these surfaces, in particular as surfaces of sensor platforms, implants and medical accessory devices. The invention also relates to the use thereof as part of coated sensor platforms, implants and medical accessory devices in addition to new organophosphoric acids and organophosphonic acids themselves.
 
     The optionally substituted compounds of formulae (IA) and (IB) wherein the B and Y groups have the above-mentioned designations, i.e. optionally substituted alkyl, alkenyl, alkinyl, aryl, aralkyl, hetaryl or hetaryl, are hereinafter referred to as organophosphoric acids and organophosphonic acids.

This application is a divisional application of Ser. No. 10/363,555,filed Mar. 5, 2003, now U.S. Pat. No. 7,517,546, which is 371application of PCT/EP01/10077, filed Aug. 31, 2001.

The invention relates to a method for precipitating mono or multiplelayers of organophosphoric acids with the general formula I (A)Y—B—OPO₃H₂  (IA)or of organophosphonic acids with the general formula I (B)Y—B—PO₃H₂  (IB)and the salts thereof, wherein B is an alkyl, alkenyl, alkinyl, aryl,aralkyl, hetaryl or hetarylalkyl residue and Y is hydrogen or afunctional group from the hydroxy, carboxy, amino, optionallylow-alkyl-substituted mono or dialkylamino series, thiol, or a negativeacid group from the ester, phosphate, phosphonate, sulfate, sulfonate,maleimide, succinimydyl, epoxy, acrylate series, wherein a biological,biochemical or synthetic recognition element may be coupled to B or Y byaddition or substitution reaction, wherein compounds may also be addedconferring the substrate surface a resistance to protein adsorptionand/or to cell adhesion and in the B chain may optionally be comprisedone or more ethylene oxide groups, rather than one or more —CH₂— groups.According to the invention, said precipitation occurs on substratesurfaces of pure or mixed oxides, nitrides or carbides of metals andsemiconductors, comprising the use of water-soluble salts of a compoundof formula (IA) or (IB) for the treatment of these surfaces, inparticular as surfaces of sensor platforms, implants and medicalaccessory devices. The invention also relates to the use thereof as partof coated sensor platforms, implants and medical accessory devices inaddition to new organophosphoric acids and organophosphonic acidstherein.

The optionally substituted compounds of formulae (IA) and (IB) whereinthe B and Y groups have the above-mentioned designations, i.e.optionally substituted alkyl, alkenyl, alkinyl, aryl, aralkyl, hetarylor hetaryl, are hereinafter referred to as organophosphoric acids andorganophosphonic acids.

For the preparation of sensor surfaces with biological or biochemicalrecognition elements immobilized on a so-called transducer for highlyefficient and highly selective binding of one or more analytes to bedetected in a sample, the nature of the transducer surface is of greatimportance. To achieve the lowest possible limits of detection, it isdesirable to immobilize in a small space as many recognition elements aspossible to which as many analyte molecules of one variety as possiblemay then be bound in the later detection process. At the same time it isdesirable on immobilization to obtain as high a degree of reactivity andbiological or biochemical functionality of the recognition elements aspossible, i.e. to minimize any signs of denaturation resulting from theimmobilization. A further objective is as far as possible to prevent thenon-specific binding or adsorption of analyte molecules which in manycases have the effect of restricting the limits of detection attainable.To fulfill these functions, a number of different methods of chemicalmodification have been developed with the aim of manufacturing so-calledbiocompatible surfaces. To some extent similar demands are made inmedicine with regard to the nature of implant surfaces.

In the field of medical implants, such as dental implants, artificialhip joints or intravascular stents, or of biomedical devices, such ascatheters or endoscopes, the nature of the surface has a significanteffect on the functionality of the part (B. Kasemo, J. Lausmaa, “SurfaceScience Aspects on Inorganic Biomaterials”, CRC Crit. Rev. Biocomp. 2(1986), 335-380). The main reason for this is the fact that, afterimplantation in the body, the surface of the implant forms the boundarybetween the biological environment and the foreign material, whereimportant processes take place, such as foreign-body reactions,inflammations, cell adhesion and formation of new tissue. Of particularimportance is the adsorption of proteins from body fluids (blood,intercellular fluid etc.), since these processes take place soon afterthe implantation and regulate the further biologically importantprocesses, in particular the accumulation of cells. The surfaceproperties of the implant are crucial here, because they directlydetermine the nature of the proteins, their strength of binding to thesurface and their conformation and orientation (B. Ratner, “New ideas inbiomaterials science—a path to engineered biomaterials”, J. Biomed. Mat.Res. 27 (1993), 837-850). These remarks are applicable to a wide varietyof applications. Examples in the field of metallic implants are:

-   1. Permanently or temporarily implanted components of steel,    cobalt-chromium alloys, titanium or titanium alloys in the skeletal    area, such as artificial hip joints, dental root implants,    osteosynthesis plates or screws. The desired objective here is to    achieve adsorption of cell-adhesive proteins which favorably    influence the integration of the part into the bone    (osteointegration).-   2. Metallic parts of steel, titanium or nickel-titanium alloys which    assume functions in contact with blood, such as stents in blood    vessels, to provide permanent prevention of arterial or venous    closure. Such parts must show a compatibility with blood which meets    the specific requirements. Often an implant surface is required here    which is resistant to protein adsorption and to platelet adhesion    and is thus a surface which shows only minimal if any tendency    towards undesirable thrombosis formation.-   3. Parts of titanium or steel which are in contact with soft tissue,    e.g. in osteosynthesis applications to support the healing of bone    fractures or in dental implants that come into contact with gingival    tissue. It is often the aim here that the soft tissue should lie    very close to the implant, but not form a firm connection therewith.

If the surfaces of such implants can be specifically adjusted to theconditions of use, the above-mentioned processes can be favorablyinfluenced and the functionality of the implant thus decisivelyimproved. There is a whole range of surface properties which have beenproven or hypothetically deduced to influence compatibility with thespecific application in the body:

-   1. The wettability of the surface or repulsion of water by the    surface (hydrophilicity/hydrophobicity of the surface, measured e.g.    as contact angle with water), which is associated with surface    energy. In this respect, it may be of advantage to set an ideal    wettability with water for a particular application.-   2. The charge of the surface (positive, negative or no charge) shows    a marked influence on the behavior of cells at the surface of an    implant, for example on that of bone cells (J. E. Davies, B.    Causton, Y. Bovell, K. Davy, C. S. Sturt, “The Migration of    Osteoblasts Over Substrata of Discrete Surface-Charge”,    Biomaterials, Vol. 7 (1986), 231-233; R. M. Shelton, I. M.    Whyte, J. E. Davies, “Interaction between Primary Bone Cell and    Biomaterials. Part 4: Colonization of Charged Polymer Surfaces”;    Biomaterials and clinical applications: proceedings of the Sixth    European Conference on Biomaterials, Bologna, Italy, Sep. 14-17,    1986; Elsevier; 1987; 597-602).-   3. The presence of functional groups or biological molecules which    are applied to the surface of the implant and, following    implantation, influence the biological process on the surface. Said    molecules include e.g. specific peptides, proteins or growth factors    (S.-J. Xiao, M. Textor, N. D. Spencer, H. Sigrist, “Covalent    Attachment of Cell-Adhesive, RGD-Containing Peptides on Titanium    Surfaces”, Langmuir 14 (1998), 5508-16, 1998).-   4. The presence of protein-repellent molecules, such as polyethylene    oxide, polyethylene glycol or heparin on the surface.

The chemical properties of the surface of commercial implants are oftennot optimized for the application to an extent that one might wish forthe medical application. The chemical properties are also often onlyincompletely controlled in commercial production. Impurities areobserved which are the consequence of processing or storage conditions.Such impurities are potentially harmful for application in the body. Inaddition they are usually not consistent from one production batch toanother or throughout the storage period and pose a risk with respect tothe quality assurance of medicinal products. Particular susceptibilityto such impurities is shown by metallic implants such as those made ofsteel or other iron alloys, or titanium and alloys thereof (TiAlV,TiAlNb, etc.), or cobalt-chromium alloys (CoCr, CoCrMo), all of whichare coated with a thin, natural oxide layer of high surface energy andthereby especially prone to contamination from the environment (e.g.through adsorption of organic components from the air, such ashydrocarbons, alcohols, etc., or through adsorption from liquids(adsorption of silicone oils, etc.).

The object of the invention is the provision of such a method for thetreatment of surfaces, especially of oxides, nitrides or carbides ofmetals or semiconductors or mixtures thereof or of oxide-coated metalsor semiconductors, using monolayers or multiple layers oforganophosphoric or organophosphonic acids or derivatives thereof asdefined hereinabove, in particular the salts thereof, in order toproduce surfaces which show a reproducible chemical composition and toadapt the properties of these surfaces for the specific application inthe field of biomaterials/implants and biosensors.

Coating compounds and methods for the antireflection and antimistcoating of surfaces are described in U.S. Pat. Nos. 5,997,621 and5,873,931. The use of a range of different amphipaths is named, buttheir combination with porous metal oxides, which are present forexample in a dispersion, only serves the formation of a network of metaloxides and amphipaths which are then applied in turn to the surface tobe coated. There are no references to a coating with amphipaths in aprocess of self-organization (formation of “self assembled monolayer”).

The application of self-assembled monolayers to metal-coated plasticfilms by means of a stamping process is described in U.S. Pat. No.5,922,550, wherein analyte-specific receptors are inserted into themonolayers to be applied. Solid substrates or other materials ascarriers for the metal coating (of gold, silver, aluminum, chrome,copper, zirconium, platinum and nickel, as well as oxides thereof) arenot described.

Not mentioned in particular is a precipitation process of theself-assembled monolayer (SAM) on a metal oxide film which would besuitable as an optical waveguide as described in a preferred embodimentof our method according to the invention. Although alkyl phosphonates orphosphonic acids are mentioned, no alkyl phosphates or alkyl phosphoricacids are mentioned.

The use of dodecylphosphate ammonium salt (DDPO₄ ammonium salt,DDPO₄(NH₄)₂ is described in a number of patents (for example U.S. Pat.Nos. 4,005,173; 4,491,531; 4,838,556; 5,873,931; 5,997,621), but not theuse thereof for the formation of a self-assembled monolayer on amacroscopic substrate through precipitation from aqueous solution.

In J. G. van Alsten, “Self-assembled monolayers on engineering metals:structure, derivatization and utility”, Langmuir 15 (1999) 7605-7614,the precipitation of SAMs based on alkyl phosphonic acids from aqueousand alcoholic solutions on so-called engineering metals (steel,aluminum, copper, and brass) is described, but not the formation of SAMsbased on alkyl phosphoric acids or derivatives thereof.

In WO 98/29580, a method is described for the treatment of metallicsurfaces of zinc, magnesium, aluminum or alloys thereof by spraying,immersion or roller-coating to improve the adhesion and corrosionresistance of lacquered and plastic-coated products. This methoddescribes alkyl phosphoric acids or alkyl phosphonic acids andderivatives thereof with 2-50 carbon atoms in the alkyl chain andvarious terminal functional groups. The precipitation occurs fromaqueous solution, wherein the solubility of some of these compounds isenhanced by the addition of organic solvents. The poorly water-solublephosphonic acids with a terminal methyl group, such as 1-phosphonic aciddodecane or 1-phosphoric acid octadecane, have not been described. Bycontrast, hydroxy-terminated molecules, such as 1-phosphoricacid-12-hydroxydodecane, have been described. The use of this class ofmolecule is confined to the above-mentioned metals, and after surfacetreatment a further compact organic coat, e.g. a lacquer or plastic, isapplied. The metal layer modified for example with alkyl phosphoric oralkyl phosphonic acids or derivatives thereof thus never serves as anouter coating exposed to the environment.

For some years, surfaces have been modified using so-calledself-assembled monolayers (hereinafter abbreviated as “SAMs”). These arevery thin, monomolecular layers which form spontaneously through thecontact of a surface with a solution of the corresponding molecule andare characterized by an organized structure of the molecule chain. Thebest-known are long-chain alkylthiols, which form SAMs of gold surfaces(R. G. Nuzzo, F. A. Fusco, D. L. Allara, “Spontaneously OrganizedMolecular Assemblies 0.3. Preparation and Properties of SolutionAdsorbed Monolayers of Organic Disulfides On Gold Surfaces”; Journal ofthe American Chemical Society, 109 (1987), 2358-2368)). Although suchgold/alkylthiol SAM surfaces are used in the biomedical field as modelsurfaces thanks to their perfectly controlled chemical surfaceproperties, they are of no interest for the manufacture of implants,because gold is only of minor importance in this field of application.

However, gold surfaces play a greater role in the field of bioanalytics.The phenomenon of surface plasmon resonance, for example, ischaracteristic of a certain type of thin metal films, in particular ofgold. In recent years, therefore, surface modification by means of SAMproduction from alkylthiols has especially been used in this field.

On the other hand, it has been shown that alkyl phosphates adsorb tooxidic surfaces. This was demonstrated for the first time on micasurfaces (J. T. Woodward et al., Langmuir 12 (1996) 6429) and onaluminum surfaces (D. L. Allara, R. G. Nuzzo, “Spontaneously OrganizedMolecular Assemblies 0.1. Formation, Dynamics, and Physical-Propertiesof Normal-Alkanoic Acids Adsorbed From Solution On an Oxidized AluminumSurface”, Langmuir 1 (1985), 45-52; D. L. Allara, R. G. Nuzzo,“Spontaneously Organized Molecular Assemblies 0.2. Quantitative InfraredSpectroscopic Determination of Equilibrium Structures ofSolution-Adsorbed Normal-Alkanoic Acids On an Oxidized AluminumSurface”, Langmuir 1 (1985), 52-66; I. Maege, E. Jaehne, A. Henke, H.-P.Adler, C. Bram, C. Jung, M. Stratmen, Macromol. Symp. 126 (1997), 7-24).The monolayers formed on mica, however, were not very stable and thus ofminimal relevance for technical applications. By contrast, it was foundthat octadecylphosphate on tantalum oxide (Ta₂O₅) forms SAMs which arestructurally very similar to those of thiols on gold and alsoconsiderably more stable than on mica (M. Textor, L. Ruiz, R. Hofer, A.Rossi, K. Feldman, G. Hähner, N. D. Spencer, “Structural Chemistry ofSelf-Assembled Monolayers of Octadecylphosphoric Acid on Tantalum OxideSurfaces” Langmuir 16 (2000), 3257-3271; D. Brovelli, G. Hähner, L.Ruiz, R. Hofer, G. Kraus, A. Waldner, J. Schlösser, P. Oroszlan, M.Ehrat, N. D. Spencer, “Highly Oriented, Self-Assembled AlkanephosphateMonolayers on Tantalum(V) Oxide Surfaces”, Langmuir 15 (1999),4324-4327). The technology of self-assembled layers ofoctadecylphosphate allows the surface of tantalum oxide to be renderedextremely hydrophobic thanks to the organized structure of thehydrophobic alkyl chains (contact angle with water: 112-115°) (FIGS. 1and 2).

Since alkyl phosphates in which B and Y have the aforementioneddesignations, for example, can in principle also be furnished withterminal groups other than methyl, e.g. with hydroxyl (—OH), amine(—NH₂) or carboxyl (—COOH), the technology naturally offers theopportunity to apply specific chemical treatments to oxide-coatedmedical implant surfaces or (optical) sensors with an oxide surface andto adapt the chemical surface properties to the application in questionand preserve its stability.

These organized alkyl phosphate layers on tantalum oxide were producedthrough contact of the oxidic surfaces with a solution of the alkylphosphate on the basis of organic solvents (heptane/isopropanol).Aqueous solutions could therefore not be used, because these long-chainalkyl phosphates are not sufficiently water-soluble.

Although short-chain alkyl phosphates would be sufficientlywater-soluble, they form almost no SAMs, because the interactionsbetween the long alkyl chains within the organized layer of theadsorbate are necessary for the formation of oriented chains andorganization.

If the surface treatment process has to be carried out in organicsolvents, problems arise which at least substantially restrict, if nothinder altogether, any commercial applications:

-   1. This type of modification is limited to the surfaces of    substrates whose materials show a sufficiently high degree of    resistance to organic solvents. For example, such a process of    surface modification for metal or metal oxide films on plastic    substrates can be virtually excluded.-   2. The use of volatile organic solvents is associated with emissions    which pose a burden on the workplace, the environment and the    atmosphere. Legislation in many countries has become very strict in    this respect and in various industries has led to such solvents    being completely supplanted by processes which cause few if any    environmental problems.-   3. If these emissions are to be eliminated, a substantial increase    in costs can be expected (closed systems, purification of exhaust    air, recovery of solvents). The resulting increase in costs often    has such a negative impact on the economic balance that such    processes become unattractive for commercial production.-   4. Specifically in the case of biomaterials and implants, organic    solvents have the serious disadvantage that they often have a    cytotoxic effect or exert a negative influence on the development of    cells and tissues. Since SAMs produced from organic solvents are    never entirely free of organic solvents, there is always a risk that    the organic volatile molecules remaining in the SAM may have a    subsequent negative effect in the body.-   5. It often has to be anticipated that the desired specific    properties cannot be achieved by a single type of molecule, but    requires a composite of SAM molecules. However, since different SAMs    have very different solubility in organic solvents (especially in    case of ω-terminal groups of differing polarity), it is often    difficult if not impossible to find a single solvent which permits    the production of a mixture of two (or more) molecules for the    precipitation of a composite SAM.

According to this invention, the aim is to eliminate the above-mentioneddisadvantages in the manufacture of SAMs, based on the organophosphatesor organophosphonates defined hereinbefore or derivatives thereof, inparticular salts, functionalized organophosphates or correspondingphosphonates, and to develop a method which allows the formation ofwell-defined SAMs on a number of metal, semiconductor, oxide, carbide ornitride surfaces without the use of solvents and the production oflayers which comprise two or more different, functionalized and/ornonfunctionalized organophosphates or -phosphonates.

The invention relates to a method for precipitating mono or multiplelayers of organophosphoric acids with the general formula I (A)Y—B—OPO₃H₂  (IA)or of organophosphonic acids with the general formula I (B)Y—B—PO₃H₂  (IB)and the salts thereof, on substrate surfaces of pure or mixed oxides,nitrides or carbides of metals and semiconductors, wherein B is analkyl, alkenyl, alkinyl, aryl, aralkyl, hetaryl or hetarylalkyl residueand Y is hydrogen or a functional group from the hydroxy, carboxy,amino, optionally low-alkyl-substituted mono or dialkylamino series,thiol, or a negative acid group from the ester, phosphate, phosphonate,sulfate, sulfonate, maleimide, succinimydyl, epoxy, acrylate series,wherein a biological, biochemical or synthetic recognition element maybe coupled to B or Y by addition or substitution reaction, whereincompounds may also be added conferring on the substrate surface aresistance to protein adsorption and/or to cell adhesion and in the Bchain may optionally be comprised one or more ethylene oxide groups,rather than one or more —CH2— groups, characterized in thatwater-soluble salts of a compound of formula (IA) or (IB) are used forthe treatment of these surfaces, in particular as surfaces of sensorplatforms, implants and medical accessory devices. The invention alsorelates to the use thereof as part of coated sensor platforms, implantsand medical accessory devices in addition to new organophosphoric acidsand organophosphonic acids themselves.

The invention especially relates to methods for precipitating mono ormultiple layers of organophosphoric acids of formula I (A) or oforganophosphonic acids of formula I (B) and salts thereof, whereingroups B and Y are combined to form an alkyl group or an optionallysubstituted alkyl group of 2-24 C atoms.

The invention relates in particular to methods for precipitating mono ormultiple layers of organophosphoric acids of formula I (A) or oforganophosphonic acids of formula I (B) and salts thereof, whereingroups B and Y are combined to form an alkyl group or an optionallysubstituted alkyl group of 2-12 C atoms.

Most particularly, depending on the intended scope of use, the inventionrelates also to methods for precipitating mono or multiple layers oforganophosphoric acids of formula I (A) or of organophosphonic acids offormula I (B) and salts thereof, wherein groups B and Y are combined toform an alkyl group or an optionally substituted alkyl group of 2-5 Catoms.

Suitable substituents are especially the substituents listed in theintroduction under Y.

The preferred layers, i.e. mono and multiple layers of organophosphoricacids of formula I (A) and organophosphonic acids of formula I (B) arealso suitable for use as part of coated sensor platforms, implants andmedical accessory devices.

Preferably compounds of formula I (A) or I (B) with various chainlengths are used, depending on the intended scope of use.

Alkyl is for example C2-C24alkyl, such as ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl,eicosanyl, heneicosanyl, docosanyl, tricosanyl or tetracosanyl. Alkenylis for example ethylene, propylene, butylene, pentylene,2-methylpentene-(1) and other higher-member alkenyls such ashexadecenyl, heptadecenyl or also octadecenyl.

Alkinyl is for example ethinyl, 2-propinyl, 2- or 3-butinyl or alsohigher-member alkinyls such as 4-pentinyl.

Aryl as such is e.g. phenyl or naphthyl, such as e.g. 1- or 2-naphthylor substituted phenyl or naphthyl, such as phenyl or naphthyl whichapart from Y are additionally substituted by lower alkyl, halogen-loweralkyl, hydroxy, lower alkoxy, lower alkanoyloxy, halogen, and/or cyano.Aryl is preferably unsubstituted phenyl or phenyl substituted asindicated above, in particular phenyl.

Arylalkyl is preferably aryl-lower alkyl, in particular phenyl-loweralkyl, quite especially phenylethyl or benzyl.

Lower alkoxy is e.g. n-propoxy, isopropoxy, n-butoxy or tert-butoxy,preferably ethoxy and in particular methoxy.

Lower alkanoyloxy is for example propionyloxy or pivaloyloxy, preferablyacetyloxy.

Halogen is for example chlorine or fluorine, in the broader sense alsobromine and iodine.

Halogen-lower alkyl is for example 2- or 3-halogen-lower alkyl, forexample 2-halopropyl, 3-halopropyl or 3-halo-2-methylpropyl.

Hetaryl is understood to mean especially a monocyclic, but also abicyclic or polycyclic residue of an aromatic character. Bicyclic andpolycyclic hetaryl may be composed of several heterocyclic rings orpreferably of one heterocyclic and one or more, e.g. one or two and inparticular one, annellated carbocyclic ring, in particular a benzo ring.Each individual ring comprises e.g. 3, 5, 6, 7 and in particular 5 or 6ring members.

Hetaryl is in particular an aza, thia oxa, thiaza, oxaza, diaza andtetrazacyclic residue.

Hetaryl is especially a monocyclic monoaza, monothia or monooxacyclicresidue, such as pyrryl, e.g. 2-pyrryl or 3-pyrryl, pyridyl, thienyl,e.g. 2- or 3-thienyl, or furyl, e.g. 2-furyl, bicyclic monoaza, monooxaor monothiacyclic residue, e.g. indolyl, e.g. 2- or 3-indolyl,quinolinyl, e.g 2- or 4 quinolinyl, isoquinolyl, 1-isoquinoline,benzofuran, e.g. 2- or 3-benzofuranyl, or benzothienyl, e.g. 2- or3-benzothienyl, monocyclic diaza, triaza, tetraza, oxaza, thiaza orthiadiazacyclic residue, such as imidazolyl, e.g. 2-imidazolyl,pyrimidinyl, e.g. 2- or 4-pyrimidinyl, triazolyl, e.g.1,2,4-triazol-3-yl, tetrazolyl, e.g. 1- or 5-tetrazolyl, oxazolyl, e.g.2-oxazolyl, isoxazolyl, e.g. 3- or 4-isoxazolyl, thiazolyl, e.g.2-thiazolyl, isothiazolyl, e.g. 3- or 4-isothiazolyl or 1,2,4- or1,3,4-thiadiazolyl, e.g. 1,2,4-thiadiazol-3-yl or 1,3,4-thiadiazol-2-yl,or bicyclic diaza, oxaza, thiazacyclic residue, such as benzimidazolyl,e.g. 2-benzimidazolyl, benzoxazolyl, e.g. 2-benzoxazolyl orbenzthiazoyl, e.g. 2-benzthiazolyl.

Hetaryl residues are unsubstituted or carry substituents as indicatedunder aryl.

Hetaryl is especially pyridyl, thienyl, pyridyl or furyl.

Hetarylalkyl residues are composed of the above-mentioned hetarylresidues and the previously named alkyl residues.

The organophosphoric acids of formula I (A) and organophosphonic acidsof formula (IB) used in the method according to the invention may formsalts with bases, e.g. corresponding alkali metal or alkaline earthmetal salts, e.g. sodium, potassium or magnesium salts, and in thebroader sense also transition metal salts, such as zinc and coppersalts, or in particular salts with ammonia or organic amines, such ascyclic amines, such as mono-, di- or tri-lower alkylamines, such ashydroxy-lower alkylamines or polyhydroxy-lower alkylamines. Cyclicamines are e.g. morpholine, thiomorpholine, piperidine or pyrrolidine.Suitable mono-lower alkylamines are for example ethyl andtert-butylamine, suitable di-lower alkylamines are for example diethyland diisopropylamine, and suitable tri-lower alkylamines are for exampletrimethyl and triethylamine, which form quaternary ammonium salts.

Appropriate hydroxy-lower alkylamines are e.g. mono-, di- andtri-ethanolamine, hydroxy-lower alkylamines are e.g. N,N-dimethyl-amino-and N,N-diethylamino ethanol. Included in the broader sense are also theabove-mentioned transition salts, which may be unsuitable for use, butmay be of advantage for the isolation or purification oforganophosphoric acids of formula I (A) or organophosphonic acids offormula I (B).

As carboxyl group, substituent Y may also form analogous basic salts.

As substituents, organophosphoric acids of general formula I (A) andorganophosphonic acids of general formula I (B) may also show a basic Ygroup.

For example, when Y is amino or optionally amino substituted by loweralkyl or di-lower alkyl. Compounds with a basic Y group may be e.g. acidaddition salts with suitable mineral acids, such as hydrogen halides,sulfuric acid or phosphoric acid, e.g. hydrochlorides, hydrobromides,sulfates, hydrogen sulfates or phosphates, salts with suitable aliphaticor aromatic sulfonic acids or N-substituted sulfamic acids, e.g.methanesulfonates, benzenesulfonates, p-toluenesulfonates orN-cyclohexylsulfaminates (cyclamates), or salts with strong organiccarboxylic acids, such as lower alkanecarboxylic acids.

Also an object of the invention are new organophoshoric acids of theformulaY—B—OPO₃H₂  (IA)and organophosphonic acids of formulaY—B—PO₃H₂  (IB)wherein B and Y have the designations indicated hereinabove with theexception of the shared designation of alkyl and salts thereof.

In particular the invention relates to new organophosphoric acids offormula I (A) and organophosphonic acids of formula I (B) wherein B andY are an aryl or arylalkyl residue and salts thereof.

The invention comprises the production of a salt of the freeorganophosphoric acid or organophosphonic acid in water, in a differentsolvent or a mixture and the simultaneous or subsequent precipitation ofthe corresponding salt by the introduction of a base which forms thecation in the resulting salt. The precipitation takes place eitherspontaneously or after a corresponding cooling or concentration of thesolution. Suitable cations are all customary positively chargedparticles, such as alkali metals (e.g. Na⁺, K⁺), the alkaline earthmetals, ammonium (NH₄ ⁺) or other quaternary ammonium ions (e.g.tetrabutylammonium). The formation of salt here takes place throughintroduction of the appropriate aqueous base (e.g. KOH or NH₄OH) intothe solution of the organophosphoric or organophosphonic acid, orthrough the introduction of a base in an organic solvent (e.g.tetrabutylammonium hydroxide in alcoholic solution) into the solution ofthe organophosphoric or organophosphonic acid, or through theintroduction of a base that is volatile (gaseous) at room temperature orat elevated temperatures (such as ammonia or a volatile amine) into thesolution of the organophosphoric or organophosphonic acid. Theresulting, saline compound then has either an inadequate solubility inthe selected solution and precipitates out spontaneously as a salt, orthe precipitation is attained by cooling or concentration of the solventvolume. It is preferred that sodium, potassium or ammonium salts inparticular be used as salts of compounds of formula (IA) and/or (IB).Especially preferred here is formation of the ammonium salt because,thanks to its low affinity for surfaces, ammonium does not interfere atall in the formation of the SAM in the subsequent SAM process.

A further advantage of these salts for their use in the method accordingto the invention is the fact that they can then be readily purified byrecrystallization and freed from unwanted impurities of the free acid.

An important characteristic of the method according to the invention isthus that the water-soluble salt of a compound of formula (IA) and/or(IB) is isolated before the precipitation of said mono or multiplelayers.

Although preparation of the ammonium salt of dodecane-phosphoric acidhas already been described (see above), it has not been described foruse as a salt in a subsequent SAM process based on an aqueous solutionof the corresponding salt.

Use of the resulting salts according to the invention relates to the useof aqueous solutions of the salt in the SAM formation process for thetreatment of oxide, carbide or nitride surfaces as specified above. As aresult, not only is it possible to produce SAMs which are free ofunwanted solvent impurities, but it is also very easy to prepare aqueousmixtures of several salts of different functionalized and/ornonfunctionalized organophosphates and/or organophosphonates.

A further important and advantageous characteristic of the methodaccording to the invention is thus that the said mono or multiple layersof compounds of formula (IA) and/or (IB) are free of organic solvents.

The preparation of SAMs from aqueous solution of (soluble)organophosphate or organophosphonate derivatives and the formation ofsodium and potassium salts of organophosphates or organophosphonatesdirectly in aqueous solution is likewise described in WO 98/29580 (seeabove). The differences from the present invention are as follows:

-   1. According to the description in WO 98/29580, the salts are not    isolated, but are simply prepared in situ. As a result, the    preferred purification option through recrystallization of the salt    before it is used for the formation of SAMs is not applicable. In    addition, this method allows a highly controlled adjustment of the    stoichiometry of the SAM solution to be used.-   2. The applications relate to completely different application    fields with completely different requirements (lacquer adhesion,    corrosion protection for lacquered products) on different substrate    materials (of zinc, aluminum, magnesium or alloys thereof).

Suitable substrates for the application of surface treatment methodsaccording to the invention are the following materials: Oxides, nitridesor carbides of tantalum, niobium, titanium, vanadium, zirconium,hafnium, molybdenum, tungsten, silicon or mixtures thereof in the formof solid bodies or as layers on substrates of any kind. Particularlysuitable are metals or metal alloys which show an oxide layer on thesurface that results either from a natural, spontaneous formation orfrom artificial production (e.g. by anodization), these being preferablythe metals titanium, tantalum, niobium, vanadium, zirconium, hafnium,molybdenum, tungsten, or silicon or alloys of these metals orsemiconductors.

Of major importance here are optically transparent oxides forapplications in the field of optical biosensors, e.g. for sensors basedon the principle of optical waveguide technology. These include inparticular, but not exclusively, metal oxides with a high refractiveindex, such as tantalum oxide (Ta₂O₅), niobium oxide (Nb₂O₅), titaniumoxide (TiO₂), zirconium oxide (ZrO₂) and mixtures of these oxides.

Particularly worthy of mention in the field of biomaterials areoxide-ceramic materials, such as aluminum oxide (Al₂O₃) and zirconiumoxide (ZrO₂). Since metallic materials with biocompatible propertiesalmost always form a natural oxide layer on the surface (passive layer,responsible for corrosion resistance and biocompatibility), the methodaccording to the invention can be advantageously applied also to anumber of implant materials. The materials to be mentioned in this fieldare titanium (with TiO₂ surface), niobium (with Nb₂O₅ surface),zirconium (with ZrO₂ surface) and tantalum (with Ta₂O₅ surface).Furthermore, the method can be applied to alloys, in particular toTi—Al—V, Ti—Al—Nb, Ti—Nb—Zr, Ti—Nb—Zr—Ta, chrome-nickel-steel (Fe—Cr—Ni,Fe—Ni—Mo), Co—Cr and Co—Cr—Mo.

Whereas essentially smooth, planar substrates are often used in sensors(but not always—capillary-based sensors are an example), the surfaceswhich are of interest in biomaterials or the implant field are oftenrough surfaces or surfaces with a topographically specific structure,because in certain applications they induce a preferred biologicalreaction in the body. Especially in metallic implants for the skeletalarea (artificial hip joints, dental implants, osteosynthesis plates andscrews) often rough or specifically roughened surfaces are used (Sittig98). Such surfaces with complex surface topography or porosityfrequently pose particular problems where the setting of specificchemical properties is concerned. The method according to the inventionfor forming SAMs based on alkyl phosphates, alkylphosphonates and thefunctionalized derivatives thereof (see also below) opens upparticularly interesting opportunities for functionalizing such complex,rough metal surfaces, because the method is equally applicable on rough,structured or porous surfaces of oxide-coated metals and metal oxides.This is explained in detail in Example 2.

The method of surface modification according to the invention is thussuitable for substrates which show an almost smooth surface with littleroughness and also for substrates which have rough surfaces, wherebythese surfaces may show an additional structuring.

It is especially preferred for biosensor applications that materials forbiochemical, biological or synthetic recognition elements (coupled to Bor Y) be selected from among the group of nucleic acids, such as DNA,RNA, oligonucleotides, nucleic acid analogs, such as PNA, monoclonal orpolyclonal antibodies, peptides, enzymes, aptamers, synthetic peptidestructures, soluble membrane-bound proteins and proteins isolated from amembrane, such as receptors, ligands thereof, antigens for antibodies,biotin, “histidine tag components” and complexing partners thereof.

It is especially preferred, however, for applications of the methodaccording to the invention for biologically compatible surfacemodification of implants that a biologically effective recognitionelement comprises peptides, proteins, glycoproteins, growth factor, suchas TGF-β, or BMP (bone morphogenic protein).

Using the method according to the invention, it is also possible toproduce strongly hydrophobic surfaces which can be passivated forexample by contact with albumins (for example, human serum albumin (HSA)or bovine serum albumin (BSA)) to achieve a protein-resistant surface.This may be important for applications for example in the field ofdevices which come into contact with blood (blood compatibility,prevention of platelet adsorption and thrombus formation).

For this purpose it may be advantageous to add to B or Y a compoundwhich confers on the substrate surface a resistance to proteinadsorption and/or cell adhesion, wherein this compound is preferablyselected from the group of compounds which are formed fromoligo(ethylene oxide), phosphoryl choline, heparin, saccharides,albumins, especially bovine serum albumin or human serum albumin,casein, nonspecific, polyclonal or monoclonal, heterologous or for theanalyte or analytes to be determined empirically nonspecific antibodies(especially for immunoassays), detergents (such as Tween 20), fragmentednatural DNA or synthetic DNA not hybridizing with polynucleotides foranalysis, such as a herring or salmon sperm extract (especially forpolynucleotide hybridization assays), or also uncharged, but hydrophilicpolymers, such as polyethylene glycols or dextrans. Groups such asoligo(ethylene oxide) show particularly good behavior in respect ofprotein resistance and have already been described with other systems,e.g. with thiol-based SAMs on gold surfaces (P. Harder, M. Grunze, R.Dahint, G. M. Whitesides, P. E. Laibinis, J. Phys. Chem. B, 102 (1998),426-436). Typical applications serve for example to improve the bloodcompatibility of stents or to prevent platelet adsorption and thrombusformation in contact with circulating blood.

It is also possible that compounds of formula (IA) and/or (IB) comprisevarious functional groups and/or biological or biochemical or syntheticrecognition elements and/or compounds for conferring on the surface aresistance to protein adsorption and/or cell adhesion in the same SAMmolecule, e.g. a protein-resistant oligo(ethylene oxide) group incombination with a biological recognition element, such as biotin.

Within the terms of the invention, it is also possible that a secondaryor further sequential monolayer might be precipitated on a primarymonolayer of organophosphates and/or organophosphonates, so that adouble or multiple layer is produced on said substrate surfaces.

A possible variant of the method comprises producing from the primarymonolayer of organophosphates and/or organophosphonates a hydrophobicsurface on which a further layer of synthetic or natural lipids isprecipitated.

In a special embodiment, the method comprises producing the saidadditional layer of synthetic or natural lipids from a lipid vesiclesuspension through spontaneous attachment of the vesicles to thehydrophobic surface of a primary monolayer of organophosphates and/ororganophosphonates, followed by distribution of the vesicle membrane onthis primary monolayer.

The synthetic or natural lipids may be selected from a group which isformed from phosphoglycerol lipids etc. or which comprises a mixture ofthese molecules. According to the embodiment described hereinbefore,molecular groups Y and/or B and/or biological or biochemical orsynthetic recognition elements and/or compounds which confer on thesurface a resistance to protein adsorption and/or cell adhesion may beassociated with the said additional layer.

A preferred embodiment of the method according to the inventioncomprises immobilizing synthetic or natural vesicles or microsomes on amultiple layer with a first monolayer of organophosphates and/ororganophosphonates on a substrate surface, optionally with associatedbiological or biochemical or synthetic recognition elements selectedfrom a group which is formed from nucleic acids (for example DNA, RNA,oligonucleotides) and nucleic acid analogs (e.g. PNA), monoclonal orpolyclonal antibodies, peptides, enzymes, aptamers, synthetic peptidestructures, soluble, membrane-bound proteins and proteins isolated froma membrane, such as receptors, ligands thereof, antigens for antibodies,biotin, “histidine tag components” and complex-forming partners thereof.

A further embodiment is the use of mixtures of various SAM molecule.Such mixed SAM layers may be produced in two ways: either by preparationof an aqueous solution which contains both SAM types, followed bytreatment of the surface in this mixture, or by sequential adsorptionusing pure SAM solutions in each case. An example of using different SAMmolecules in aqueous solution to form mixed SAM systems on the surfaceis precipitation from a mixture of SAM molecules which comprise abiological recognition element such as biotin and SAM molecules whichcomprise a group such as oligo(ethyleneoxide) to produce a surface witha protein-resistant background in the simultaneous presence of abiologically specific function (biotin).

SAMs are described as mixed when they comprise two molecules in anyratio, wherein the above-mentioned classes of molecules may berepresented in any combination.

A further object of the invention therefore relates to a method forprecipitating mixed mono or multiple layers of organophosphoric acids ofthe general formula I (A)Y—B—OPO₃H₂  (IA)and/or of organophosphonic acids of the general formula I (B)Y—B—PO₃H₂  (IB)and the salts thereof, on substrate surfaces of pure or mixed oxides,nitrides or carbides of metals and semiconductors, wherein B is analkyl, alkenyl, alkinyl, aryl, aralkyl, hetaryl or hetarylalkyl residueand Y is hydrogen or a functional group from the hydroxy, carboxy,amino, optionally low-alkyl-substituted mono or dialkylamino series,thiol, or a negative acid group from the ester, phosphate, phosphonate,sulfate, sulfonate, maleimide, succinimydyl, epoxy, acrylate series,wherein a biological, biochemical or synthetic recognition element maybe coupled to B or Y by addition or substitution reaction, whereincompounds may also be added conferring on the substrate surface aresistance to protein adsorption and/or to cell adhesion and in the Bchain may optionally be comprised one or more ethylene oxide groups,rather than one or more —CH2— groups, characterized in thatwater-soluble salts of a compound of formula (IA) or (IB) are used forthe treatment of these surfaces, in particular as surfaces of sensorplatforms, implants and medical accessory devices.

It is preferred in turn that before precipitation of said mixedmonolayers or multiple layers the water-soluble salt of a compound offormula (IA) and/or (IB) is isolated.

Also in the case of mixed SAMs the method according to the inventioncomprises the said monolayers and multiple layers of compounds offormula (IA) and/or (IB) being free of organic solvents.

Here too it is preferred that sodium, potassium or ammonium salts inparticular be used as salts of compounds of formula (IA) and/or (IB).

Also suitable for this embodiment of the method according to theinvention are oxides, nitrides or carbides of tantalum, niobium,titanium, vanadium, zirconium, hafnium, molybdenum, tungsten, silicon ormixtures thereof in the form of solid bodies or as layers on substratesof any kind.

A preferred embodiment of the method according to the invention forprecipitating mixed monolayers or multiple layers comprises using forthe coating compounds of formula (IA) and/or (IB), wherein the B and Ygroups are both an alkyl group or an optionally substituted alkyl groupof 2-24 C atoms.

It is in turn especially preferred for biosensor applications thatmaterials for biochemical, biological or synthetic recognition elements(coupled to B or Y) be selected from among the group of nucleic acids,such as DNA, RNA, oligonucleotides, nucleic acid analogs, such as PNA,monoclonal or polyclonal antibodies, peptides, enzymes, aptamers,synthetic peptide structures, soluble membrane-bound proteins andproteins isolated from a membrane, such as receptors, ligands thereof,antigens for antibodies, biotin, “histidine tag components” andcomplexing partners thereof.

It is especially preferred, however, also for applications of the methodaccording to the invention for biologically compatible surfacemodification of implants that a biologically effective recognitionelement comprises peptides, proteins, glycoproteins, growth factor, suchas TGF-β, or BMP (bone morphogenic protein).

Using the method according to the invention for precipitating mixedmonolayers or multiple layers, it is also possible to produce stronglyhydrophobic surfaces which can be passivated for example by contact withalbumins (for example, human serum albumin (HSA) or bovine serum albumin(BSA)) to achieve a protein-resistant surface.

For this purpose it may in turn be advantageous to add to B or Y acompound which confers on the substrate surface a resistance to proteinadsorption and/or cell adhesion, wherein this compound is preferablyselected from the group of compounds which are formed fromoligo(ethylene oxide), phosphoryl choline, heparin, saccharides,albumins, especially bovine serum albumin or human serum albumin,casein, nonspecific, polyclonal or monoclonal, heterologous or for theanalyte or analytes to be determined empirically nonspecific antibodies(especially for immunoassays), detergents (such as Tween 20), fragmentednatural DNA or synthetic DNA not hybridizing with polynucleotides foranalysis, such as a herring or salmon sperm extract (especially forpolynucleotide hybridization assays), or also uncharged, but hydrophilicpolymers, such as polyethylene glycols or dextrans.

It is also possible that compounds of formula (IA) and/or (IB) comprisevarious functional groups and/or biological or biochemical or syntheticrecognition elements and/or compounds for conferring on the surface aresistance to protein adsorption and/or cell adhesion in the same SAMmolecule.

The substrates may in turn show a smooth surface with low roughness orpossess rough surfaces, wherein these surfaces may optionally showadditional structuring.

A particular characteristic of the method according to the invention forproducing mixed SAMs is that is creates the opportunity to control thehydrophilicity or hydrophobicity of the surface by selecting the ratioof the mixture. This opens up the possibility of a controlled adjustmentof surface wettability (contact angle with water). Wettability is animportant characteristic in the field of biocompatibility. For certainapplications it may be advantageous to set a medium contact angle. As anexample of this, the production and characterization of a mixed SAM fromdodecyl phosphate/co-hydroxy-dodecyl phosphate is described hereinbelow(see Example 1).

A further characteristic is that a controlled density of positive and/ornegative charges on the surface can be achieved by selecting the ratioof the mixture. Surface charge plays an important role with regard tothe interaction with biological cells (J. E. Davies, B. Causton, Y.Bovell, K. Davy, C. S. Sturt, “The Migration of Osteoblasts OverSubstrata of Discrete Surface-Charge”, Biomaterials, Vol. 7 (1986),231-233; R. M. Shelton, I. M. Whyte, J. E. Davies, “Interaction betweenPrimary Bone Cell and Biomaterials. Part 4: Colonization of ChargedPolymer Surfaces”, Biomaterials and clinical applications: proceedingsof the Sixth European Conference on Biomaterials, Bologna, Italy, Sep.14-17, 1986; Elsevier (1987), 597-602). Suitable systems are, forexample: Organophosphates or organophosphonates with a terminal aminegroup (positively charged at a body pH of 7.4) or organophosphates ororganophosphonates with a co-terminal negatively charged chemicallyfunctional group, such as phosphate or phosphonate, sulfate orsulfonate, carboxylate, etc.

The method according to the invention also enables a controlled densityof reactive groups and/or biochemical recognition elements or biological“functions” to be achieved by selecting the ratio of the mixture.

Since selective adsorption properties are observed when using aqueoussolutions of organophosphates or organophosphonates (see Example 1),this method permits the production of surfaces which are coated onlylocally with the SAM while other zones of the surface remain uncoated tobe manufactured in a single step.

A further object of the invention is therefore to produce chemicallystructured surfaces by local precipitation of monolayers or multiplelayers of organophosphates and/or organophosphonates on substratesurfaces of pure or mixed oxides, nitrides or carbides of metals orsemiconductors, comprising the use of aqueous saliniform compounds ofthe corresponding organophosphoric acid or organophosphonic acid for thetreatment of surfaces and precipitating the pure or mixed monolayers ormultiple layers using one of the embodiments of the method mentionedhereinbefore.

In particular, silicon dioxide shows almost no tendency for adsorptionof organophosphates or organophosphonates from aqueous solution.Chemically structured surfaces, such as can be manufactured e.g bylithographic or other mask techniques, may thus be used for specificproduction of surfaces which show specifically differing chemicalproperties (see Example 1). This technique is useful both for thechemical structuring of biomaterial or implant surfaces, in order e.g.to achieve local control of the adsorption of proteins and growthfactors or the adhesion of cells and thus exert an influence on thespecific response of the biological environment (in vitro or in vivo) onthe surface of the foreign material, and also for structuring thesurfaces of biosensors with corresponding locally modifiable properties.

A preferred variant of the method comprises the substrate surfaceshowing a defined pattern with silicon dioxide or transition metaloxides. A further development of this variant comprises a furtherprecipitation of mono- or multiple layers of organophosphates and/ororganophosphonates from organic solvents on the silicon dioxide areas.

Finally, the technique is suitable for the partial coating of parts.This is of interest e.g. in the field of medical implants, wherediffering requirements are often made on different areas of the implant.For example, dental root implants show zones which come into contactwith bony tissue following implantation, whereas other zones of the sameimplant come into contact with the gingiva. These two zones may bemodified with the method according to the invention in such a way thatthey show a surface composition optimized for the local requirementsprofile. The different SAM layers may be locally and selectivelyapplied, e.g. by partial immersion, brushing, spreading, imprinting orby inkjet methods.

For application of the mono or multiple layers, especially on planarsubstrates, there are numerous methods available. It is preferred thatthe mono or multiple layers are precipitated, optionally in a localselective manner, using a method selected from the group comprisingimmersion, spreading, brushing, “inkjet spotting”, mechanical spottingby means of a stylus, pen or capillary, “micro-contact printing”,fluidic contact of the substrate surface with parallel or crossedmicrochannels, under the influence of pressure differences or electricalor electromagnetic potentials.

A special embodiment of the method according to the invention forproducing chemically structured surfaces comprises local hydrophilic orhydrophobic areas being produced by local precipitation of mono ormultiple layers of organophosphates and/or organophosphonates onsubstrate surfaces of pure or mixed oxides, nitrides or carbides ofmetals or semiconductors and their surrounding substrate surface thenbeing coated with a terminally hydrophobic or hydrophilic monolayer.

A further development comprises one or more monolayers being locallyprecipitated as described hereinbefore on a chemically structuredsurface produced by the method according to the invention and double ormultiple layers thus being locally produced.

As explained hereinbefore, the precipitation method according to theinvention is especially suitable for manufacturing substrate surfaces intwo different fields of application. A preferred object of the inventionrelates to the manufacture of implant surfaces with implants fromoxide-coated metals, such as titanium, tantalum, niobium, alloys such astitanium-aluminum-vanadium, titanium-aluminum-niobium,titanium-niobium-zirconium, titanium-niobium-zirconium-tantalum,cobalt-chromium, cobalt-chromium-molybdenum, iron-nickel-chromium,wherein pure or mixed mono or multiple layers of organophosphates and/ororganophosphonates are precipitated on the surface according to one ofthe embodiments mentioned hereinbefore.

A further object of the invention is a method for manufacturing sensorplatform surfaces comprising the precipitation of pure or mixed mono ormultiple layers of organophosphates and/or organophosphonates on thesurface according to one of the embodiments mentioned hereinbefore.

The invention also encompasses an implant with a mono or multiple layerof organophosphates and/or organophosphonates as surface, comprising theproduction of the surface using a precipitation method according to oneof the embodiments mentioned hereinbefore.

The implant according to the invention may be selected from the group ofroot implants for dental applications, artificial prostheses, such aship joint stems, balls and sockets, artificial knee joints,osteosynthesis components, such as bone plates, screws, “fixateurexteme”, components for the repair of damage in the cranial region(“maxillofacial devices”), components in the field of spinal surgery(“spinal surgery implants”), stents, and cardiac pacemaker components.

Further objects of the invention are medical accessory devices of metalsor ceramic with a mono or multiple layer of organophosphates and/ororganophosphonates as surface, comprising the production of the surfaceusing a precipitation method according to one of the embodimentsmentioned hereinbefore.

Medical accessory devices of metal or ceramic according to the inventionmay be selected from the group comprising catheters, balloon catheters,endoscopes, components for exogenous, blood-carrying systems, such ascardiovascular machines.

A further object of the invention is a sensor platform with a mono ormultiple layer of organophosphates and/or organophosphonates as surface,comprising the production of the surface using a precipitation methodaccording to one of the embodiments mentioned hereinbefore.

The sensory platform preferably comprises at least one array ofbiological or biochemical or synthetic recognition elements, immobilizedin discrete measurement areas, for the specific recognition and/orbinding of one or more analytes and/or specific interaction with saidanalytes.

Numerous possible embodiments of the sensor platform according to theinvention comprise the detection of one or more analytes by means oflabels selected from the group formed from e.g. luminescence labels,especially luminescent intercalators or “molecular beacons”, absorptionlabels, mass labels, especially metal colloids or plastic beads, spinlabels, such as ESR or NMR labels, or radioactive labels.

A possible variant comprises detection of the analyte based on thedetermination of a change in the effective refractive index as a resultof molecular adsorption or desorption on the measurement areas.

A sub-variant comprises the detection of analyte based on thedetermination of a change in the conditions for generating a surfaceplasmon in the metal layer of a multiple layer system, wherein the metallayer preferably comprises gold or silver.

A preferred embodiment of the sensor platform according to the inventioncomprises the detection of analyte based on the determination of achange in one or more luminescences.

A possible variant comprises delivering the excitation light in avertical illuminator.

Depending on the specific embodiment, variants are preferred whichcomprise the material of the sensor platform that is in contact with themeasurement areas being transparent or absorbent within a depth of atleast 200 nm from the measurement areas in at least one excitationwavelength.

Another possible embodiment is designed in such a way that theexcitation light is delivered in a transmission configuration. For thisembodiment, the material of the sensor platform has to be transparent inat least one excitation wavelength.

A preferred embodiment of the sensor platform according to the inventioncomprises the sensor platform being formed as an optical waveguide whichis preferably essentially planar.

The sensor platform is preferably an optically transparent material fromthe group comprising silicates, e.g. glass or quartz, transparentthermoplastic or moldable plastic, for example polycarbonate, polyimide,acrylates, especially polymethylmethacrylate, or polystyrene.

An especially preferred embodiment of the sensor platform according tothe invention comprises an optical thin-layer waveguide with a layer (a)which is transparent in at least one excitation wavelength on a layer(b) which is likewise transparent in at least this excitation wavelengthwith a lower refractive index than layer (a).

Various embodiments of such sensor platforms and methods for thedetection of one or more analytes using such sensor platforms aredescribed in detail for example in patents U.S. Pat. Nos. 5,822,472,5,959,292 and 6,078,705 as well as in patent applications WO 96/35940,WO 97/37211, WO 98/08077, WO 99/58963, PCT/EP 00/04869 and PCT/EP00/07529. The herein described embodiments of sensor platforms whosesurface has been modified according to the method of the invention andof methods for the detection of one or more analytes using such modifiedsensor platforms are likewise the object of the present invention.

A further object of the invention is a method for the simultaneousqualitative and/or quantitative detection of numerous analytescomprising one or more liquid samples to be tested for said analytesbeing brought into contact with the measurement areas on a sensorplatform according to the invention and the resulting changes in signalsfrom the measurement areas being measured.

The detection of analytes is preferably based on determining the changein one or more luminescences.

A possible embodiment of the method according to the invention comprisesdelivering the excitation light from one or more excitation lightsources in a vertical illuminator.

Another possible embodiment comprises delivering the excitation lightfrom one or more excitation light sources in a transmissionconfiguration.

The sensor platform is preferably formed as an optical waveguide whichis preferably essentially planar, and the excitation light from one ormore light sources is preferably coupled into the optical waveguideusing a method selected from the group comprising butt joint coupling,coupling via suitable optic fibers as optical waveguides, prismcoupling, grating coupling or evanescent coupling by overlapping of theevanescent field of said optical waveguide with the evanescent field ofa further waveguide brought into near-field contact therewith.

The addition of one or more samples and of the detection reagents to beused in the method of detection may take place sequentially in severalsteps. One or more samples are preferably incubated beforehand with amixture of the various detection reagents for determining the analytesto be detected in said samples and these mixtures then added in a singlestep to the arrays set up for this purpose on the sensor platform.

A further object of an embodiment of the method according to theinvention is the calibration of luminescences generated by the couplingof one or more analytes or by the specific interaction with one or moreanalytes in the near field of layer (a) comprising the addition of oneor more calibration solutions with known concentrations of said analytesto be determined to the same or different measurement areas or segmentsof measurement areas or arrays of measurement areas on a sensor platformto which one or more of the samples to be tested are added in the sameor in a separate step.

An object of the invention is a method according to one of theembodiments mentioned hereinbefore for simultaneous or sequential,quantitative or qualitative determination of one or more analytes fromthe group of antibodies or antigens, receptors or ligands, chelators or“histidine tag components”, oligonucleotides, DNA or RNA strands, DNA orRNA analogs, enzymes, enzyme cofactors or inhibitors, lectins andcarbohydrates.

Possible embodiments of the method comprise the samples to be testedbeing naturally occurring body fluids such as blood, serum, plasma,lymph or urine or egg yolk or optically turbid fluids or tissue fluidsor surface water or soil or plant extracts or biological or syntheticprocess broths or being taken from biological tissue parts or from cellcultures or extracts.

A further object of the invention is the use of a sensor platformaccording to the invention and/or an analytical system according to theinvention and/or a method according to the invention for quantitative orqualitative analysis for the determination of chemical, biochemical orbiological analytes during screening procedures in pharmaceuticalresearch, combinatorial chemistry, clinical and preclinical development,for real-time binding studies and for the determination of kineticparameters in affinity screening and in research, for qualitative andquantitative determination of analytes, especially for DNA and RNAanalysis, for the performance of toxicity studies, and for thedetermination of gene or protein expression profiles, as well as for thedetection of antibodies, antigens, pathogens or bacteria inpharmaceutical product development and research, human and veterinarydiagnosis, agrochemical product development and research, symptomaticand presymptomatic crop diagnosis, for patient stratification inpharmaceutical product development and for the therapeutic selection ofmedicines, for the detection of pathogens, noxae and germs, especiallysalmonellae, prions, viruses and bacteria, in food analysis andenvironmental analysis.

Embodiments of the invention are illustrated in the following examples.

EXAMPLE 1 Manufacture of Dodecylphosphate SAM andHydroxydodecylphosphate SAM as Well as Mixed SAMs on Metal OxideSubstrates from Aqueous Solution of the Corresponding Ammonium Salts

1.1 Objective and Specification

The aim of this example is to show that the use of ammonium salts ofalkyl phosphates in aqueous solution according to the invention permitsthe specific modification of various oxidic surfaces. The example of theammonium salt of hydroxy-terminated dodecylphosphoric acid further showsthat the invention also permits the production of well-defined SAMs withterminally functionalized alkyl phosphates. It is further shown that theuse of mixtures of different alkyl phosphates permits the specific and“stepless” fine adjustment of surface conditions, which is of decisiveimportance for functionalizing the surfaces both of sensor platforms(“biosensor chips”) and of implants and for the functionality thereof.

1.2 Materials and Methods

1.2.1 Substrates

Ta₂O₅, Nb₂O₅: small glass plates (15×15×1 mm) were coated with a 150-nmthick layer of Ta₂O₅ or Nb₂O₅ (Balzers AG, Balzers, Liechtenstein).

Ti_(0.4)Si_(0.6)O₂, Fe₂O₃, ZrO₂, SiO₂: AF45 glass substrates (8×12×1 mm)were coated with a 200-nm thick Ti_(0.4)Si_(0.6)O₂ coat. For the testson Fe₂O₃, ZrO₂ and SiO₂ surfaces, an additional layer (thickness: 14 nm)of the corresponding metal oxide was deposited as outermost layer(Microvacuum, Ltd., Budapest, Hungary).

TiO₂: glass substrates were coated by sputtering with a 100 nm thicklayer of TiO₂ (Paul Scherrer Institute, Villigen, Switzerland).

Al₂O₃: Al samples (99.9% purity) of 1 mm thickness were anodized at 25 Vin a junction electrolyte, which leads to an oxide layer thickness ofabout 30 nm (Alusuisse Technology Center, Neuhausen am Rheinfall,Switzerland).

1.2.2 Alkyl Phosphates

a) DDPO₄(NH₄)₂:

Precipitation of Ammonium Salt

2.00 g dodecylphosphate (DDPO₄) (technical quality, Aldrich) wasdissolved in 200 ml of 2-propanol (UVASOL, Merck), heated to 82° C. andboiled under reflux. Then 6 ml of ammonia (25% aq., reagent grade,Merck) was added. After cooling in ice water, the precipitated ammoniumsalt of DDP was filtered, washed with ice water and dried at 60° C. and10 mbar vacuum for 20 h. 61 g of a white powder (m.p: 225°) wasisolated, which corresponds to a yield of 71%.

Control by Means of ¹H-NMR

(DMSO or CD₃OD): 0.88 ppm (t, 3H, —(CH₂)_(n)CH₃), 1.28 ppm (m, 18H,—CH₂CH₂(CH₂)₉CH₃), 1.54 ppm (m, 2H, —CH₂CH₂(CH₂)₉CH₃), 3.72 ppm (q, 2H,—OCH₂CH₂(CH₂)₉CH₃), 4.9 (s, 8H, NH₄). The ³¹P-NMR comprises a singlepeak, which suggests a pure compound.

Elemental Analysis

Calculated as monoammonium salt: [C] 50.87%, [H] 10.67%, [N] 4.94% [O]22.59%, [P] 10.93%. Experimental analysis: [C] 50.61%, [H] 10.94%, [N]4.95% [O] 22.75%, [P] 10.69%.

b) OH-DDPO₄(NH₄)₂:

Precipitation of Ammonium Salt

500 mg of hydroxydodecylphosphate (OH-DDPO₄) was dissolved in 20 ml of2-propanol (UVASOL, Merck), and the solution was purged with NH₃ gas for5 minutes. The precipitated ammonium salt of OH-DDPO₄ was isolated bycentrifugation, washed and dried in a stream of dry nitrogen gas. 515 mgof white powder was isolated (yield: 91%).

Control by Means of ¹H-NMR

(DMSO or CD₃OD): 1.24 ppm (m, 16H, —CH₂CH₂(CH₂)₈CH₂CH₂OH), 1.38 ppm (m,2H, —CH₂CH₂(CH₂)₈CH₂CH₂OH), 1.44 ppm (m, 2H, —CH₂CH₂(CH₂)₁₀OH), 3.35 ppm(t, 2H, —(CH₂)_(n)CH₂OH), 3.57 ppm (q, 2H, —OCH₂CH₂(CH₂)₁₀OH), 5.2 (s,8H, NH₄).

Elemental Analysis

Calculated as diammonium salt: [C] 45.6%, [H] 10.5%, [N] 8.9%.Experimental analysis: [C] 44.1%, [H] 9.6%, [N] 5.7%.

The elemental analysis shows that, in comparison with the diammoniumsalt, the nitrogen content is somewhat lower than expected. This isprobably attributable to a certain loss of ammonia and conversion to themonoammonium salt. It can be concluded from the NMR spectra, which donot contain any nonidentifiable peaks, that there are no impurities inhigh concentrations.

1.2.3 Preparation of the Treatment Solution

-   A) 150 mg of DDPO₄(NH₄)₂ was dissolved in 5 ml of ultrapure water,    the solution being gently heated to 50° C. The volume was made up to    100 ml.-   B) 158 mg of OH-DDPO₄(NH₄)₂ is dissolved in 5 ml of ultrapure water    by heating to about 80° C. The solution was passed through a 0.22 μm    filter (MILLEX-GV, MILLIPORE, Bedford, Mass.) and the volume made up    to 100 ml.

The two solutions a) and b) were mixed in various ratios, from 0 to 100%by volume in relation to OH-DDPO₄(NH₄)₂. 11 different solutions, whosecontent of OH-DDPO₄(NH₄)₂ was increased in each case by 10%, wereprepared. The total content of phosphate was maintained at a constant0.5 mM.

1.2.4 Sample Treatment

The substrates for the surface modification tests were cleaned uponsonication for 15 min in ultrapure water and then for 15 min in2-propanol. After drying, they were subjected for 3 min to oxygen-plasmacleaning (Harrick Plasma Cleaner/Sterilizer PDC-32G, Ossining, N.Y.,USA). They were then transferred to a glass vessel, to which the aqueoussolution of alkyl phosphate was then added. After treatment for 48 h,the samples were taken, rinsed with water and dried in a stream ofnitrogen.

1.3 Results

a) DDPO₄ on Various Substrates

The alkyl phosphates were applied to the following surfaces: Al₂O₃,Ta₂O₅, Nb₂O₅, ZrO₂, Fe₂O₃, TiO₂, Ti_(0.4)Si_(0.6)O₂ and SiO₂. Treatmentwas carried out in 0.5 mM DDPO₄(NH₄)₂, as described in the previoussection.

1. Contact Angle

The advancing contact angles are summarized in Table 1. Results showthat highly hydrophobic, self-assembled monolayers are formed on allmetal oxides. A contact angle of greater or equal 110° is typical ofperfect SAMs. Exceptions are silicon dioxide (SiO₂) and TiO_(0.6)SiO₄O₂.The isolelectric point does not play any visible role here; it variesfrom 2.7-3.0 for Ta₂O₅ to 7.0-8.6 for Fe₂O₃ (Table 1). On theSiO₂-surface the contact angle remains within the limits of the valuesprior to treatment, i.e. the surface remains completely hydrophilic. Itis thus clear that this surface does not react with the alkyl phosphatesand does not form a SAM.

The Ti_(0.4)Si_(0.6)O₂ layer on the glass chips comprises aheterogeneous structure of TiO₂ and SiO₂. The TiO_(0.4)Si_(0.6)O₂surfaces form an incomplete DSAM in the treatment solution, wherein thecontact increases, but only values of about 64° are reached. Thiscorrelates with the observation that while SAMs are formed on TiO₂, theyare not formed on SiO₂.

TABLE 1 Literature values of isoelectric points (IEP) of metal oxidesused, experimentally measured contact angle (CA) after treatment withaqueous DDPO₄(NH₄)₂ solution, and droplet density (DD) measured afterformation of the condensation figures on the same surfaces treated withDDPO₄(NH₄)₂; (SD = standard deviation). Substrates IEP Ref (IEP) CA (±SD) DD (± SD) Ta₂O₅ 2.7-3.0 14 114.6 ± 0.48 129 ± 19 Al₂O₃ 7.5-8.0 15111.4 ± 1.07 152 ± 22 Nb₂O₅ 3.4-3.6 16 109.7 ± 0.63 120 ± 18 ZrO₂ 4.0 17110.1 ± 0.61 258 ± 39 Fe₂O₃ 7.0-8.6 18/19 110.8 ± 0.64 197 ± 30Ti_(0.4)Si_(0.6)O₂ 3.6 20  63.8 ± 1.93 334 ± 50 TiO₂ 4.7-6.2 21 111.4 ±1.18 221 ± 33 SiO₂ 1.8-2.2 22  10.0 ± 3.42 >30002. Droplet Density

DDPO₄ SAMs on the pure metal oxides show, after formation of thecondensation film, a very low droplet density of 120-260 droplets/mm²(Table 1) and a homogeneous distribution of droplets over the surface.SiO₂ remains hydrophilic, and a continuous water film is formed, becausethe high density of droplets rapidly leads to coalescence. On theTi_(0.4)Si_(0.6)O₂ surface coated with DDPO₄, a DD of about 350dropöets/mm² forms. This value, as expected, is higher than on the puremetal oxide surfaces which form SAMs and suggests an incomplete SAMformation.

By means of precipitation from the gas phase, Fe₂O₃ was applied in theform of 2 mm wide strips to glass chips coated with Ti_(0.4)Si_(0.6)O₂.During the rinsing process after coating in the DDPO₄ solution it becameobvious that only the Fe₂O₃ zones became hydrophobic, whereas theadjacent Ti_(0.4)Si_(0.6)O₂ zones remained hydrophilic. The observed 2mm wide water-repellent strips in FIG. 4 correspond to the Fe₂O₃ zone.

3. X-Ray Photoelectron Spectroscopy

After immersion of the metal oxide and silicon dioxide sample surfacesin a 0.5 mM aqueous DDPO₄(NH₄)₂ solution, the surfaces were analyzed byx-ray photoelectron spectroscopy (XPS) in two different exit angles. Atan electron exit angle of 15° (in relation to the surface) the XPSanalysis is highly surface-sensitive, whereas at an angle of 75° deeperzones (substrate) are also included in the analysis.

The concentrations of C, O, P and of the corresponding metal oxidecations were quantified by means of standard sensitivity factors (Table2a and b).

The quantity of the SAM analyzed lies in the same range for all metaloxides studied and suggests a monolayer surface occupancy. In conformitywith the measurements of contact angle and droplet density, no SAMs wereobserved on the SiO₂ samples (no phosphorus signal and comparativelylittle carbon). On the mixed layer (Ti_(0.4)Si_(0.6)O₂) about half asmuch titanium is detected as on the pure titanium dioxide (TiO₂) layer.Accordingly, only about half the concentrations of P and C are foundcompared with the corresponding TiO₂ surface. This in turn demonstratesthat the SAM only forms on TiO₂, but not on SiO₂.

TABLES 2a and b Atomic concentrations measured by XPS of metal oxidesubstrates coated with DDPO₄. Electron exit angle: 15° and 75° inrelation to the surface. The values in parentheses correspond to theatomic concentrations of titanium from the substrate under thesputter-coated Fe₂O₃ sample, or to the atomic concentration of siliconin the case of the Ti_(0.4)Si_(0.6)O₂ sample. Substrate MO_(x) % C % O(tot) % M % P Atomic concentrations (15° exit angle) Ta₂O₅ 67.8 23.26.71 2.36 Al₂O₃ 67.6 21.1 8.85 2.53 Nb₂O₅ 59.7 29.6 8.5 2.26 ZrO₂ 72.222.0 3.06 2.77 Fe₂O₃ 66.1 27.5 Fe: 3.09/Ti: 0.41 2.86 TiO₂ 68.6 23.06.13 2.23 SiO₂ 6.2 70.8 22.9 0 Ti_(0.4)Si_(0.6)O₂ 26.2 54.5 Ti: 3.04/Si:15.1 1.13 Atomic concentration (75° exit angle) Ta₂O₅ 27.3 53.7 17.41.61 Al₂O₃ 25.3 47.1 25.5 2.10 Nb₂O₅ 31.9 49.4 17.3 1.30 ZrO₂ 35.9 49.012.9 2.28 Fe₂O₃ 28.9 56.5 Fe: 10.2/Ti: 2.46 1.93 TiO₂ 31.2 49.4 17.61.83 SiO₂ n.b.^(a)) n.b.^(a)) n.b.^(a)) n.b.^(a)) Ti_(0.4)Si_(0.6)O₂14.9 60.2 Ti: 9.97/Si: 14.1 0.85 ^(a))not observedb) OH-DDPO₄ on Ta₂O₅ and Nb₂O₅

Self-assembled layers of 12-hydroxydodecylphosphate (OH-DDPO₄) onsamples coated with Ta₂O₅ and Nb₂O₅ were prepared by immersion in anaqueous solution of 0.5 mM OH-DDPO₄(NH₄)₂, as described in theexperimental section (see above).

1. Contact Angle and XPS

The advancing contact angle was measured immediately after surfacetreatment of the samples. It amounts to about 50°, i.e. the surface isstrongly hydrophilic compared with the methyl-terminated layer of DDPO₄,but less hydrophilic than the cleaned metal oxide surface (contact angle<10°).

This is a clear indication that the hydroxy groups on the surface areactually exposed. To prove this, XPS spectra were measured at differentexit angles. The variation of signal intensity as a function of exitangle enables evidence to be obtained on the position of thecorresponding element. The O(1s) signal is shown in FIG. 5 for the twodifferent exit angles 11.5° and 20.5°. Compared with the pure DDPO₄layer, the oxygen signal (O1s) shows an additional shoulder at a bindingenergy of 533.4 eV (FIG. 5), the intensity being significantly higher at11.5° than at 20.5°. The position of the O(1s) signal here is typical ofhydroxy functions.

No nitrogen was detected, indicating that the ammonium cation does notparticipate in the formation of the SAM on the surfaces studied here.

It has already been shown that the water contact angle correlates withthe level of SAM occupancy. Contact angles of hydroxy-terminated SAMswere measured at 50-80°. To test whether hydroxy-terminated alkylphosphates form a densely packed layer similar to DDPO₄, the XPS C1sintensities of the DDPO₄ SAMs were compared with those of the OH-DDPO₄SAMs at different exit angles (FIG. 6). The results show that thecorresponding C1s signals are closely comparable. This demonstrates thathydroxy-terminated alkyl phosphate (OH-DDPO₄) forms layers also formsdensely packed layers as those they are typical of organized SAMs.

The atomic concentrations of C, O, P and of the substrate cation Ta orNb were calculated from the corresponding XPS intensities (Table 3). Thedata are consistent with the model of a surface on which the phosphatesare bound to the metal oxide, whereas the terminal groups (hydroxy andmethyl) point away from the surface (“tail-up” configuration).

The O1s oxygen signals were deconvoluted into three components: a) themetal oxide oxygen (530.2 eV), by the phosphate oxygen (531.4 eV for P—Ometal and P═O and 532.6 eV for R—O—P and P—OH), and c) the hydroxideoxygen (OH) at 533.4 eV. This assignment is based on a correspondingdetailed analysis of the XPS spectra. The data confirm that not onlypure alkyl phosphate, but also OH-DDPO₄ binds to the surface in analigned manner (FIG. 7).

The increase in the oxygen signal of the metal oxide substrate with theincreasing angle of detection is caused by the increasing informationdepth of the method (maximum analyzed depth at perpendicular (90°)detection). The oxygen signal of the phosphate group decreases slightlywith an increasing angle of detection. This is typical of elementslocated at the boundary surface. The oxygen signal of the hydroxy groupincreases slightly with a decreasing angle of detection, which istypical of a hydroxy group position at the boundary surface of SAM/airor vacuum.

TABLES 3a and b XPS atomic concentrations of self-assembling OH-DDPO₄layers on Ta₂O₅ and Nb₂O₅ as a function of the angle of detection Θused. The oxygen signal was deconvoluted into three components. Angle ofAtom-% Atom-% Atom-% Atom-% detection Θ Sin Θ Ta % O % P % C 11.5 0.203.05 26.6 3.69 66.72 20.5 0.35 6.27 33.0 3.91 56.79 30.0 0.50 9.21 39.83.12 47.78 40.5 0.65 11.9 45.8 2.83 39.46 53.1 0.80 13.42 50.3 2.0034.29 71.8 0.95 14.88 53.3 2.12 29.65 Angle of Atom-% detection Θ Sin ΘNb Atom-% O Atom-% P Atom-% C 11.5 0.20 4.64 29.3 3.86 62.21 20.5 0.356.74 33.9 3.32 55.97 30.0 0.50 9.53 38.5 3.07 48.62 40.5 0.65 11.97 43.62.78 41.97 53.1 0.80 14.05 47.7 2.01 36.24 71.8 0.95 16.38 51.6 2.0729.98c) Mixed SAMs of OH-DDPO₄/DDPO₄ on Ta₂O₅ and TiO₂

Mixtures of aqueous solutions of 0.5 mM OH-DDPO₄(NH₄)₂ and 0.5 mMDDPO₄(NH₄)₂ were prepared as described in the experimental section.Glass chips coated with Ta₂O₅ were cleaned and treated in the solutionsas described in the experimental section.

1. Contact Angle and Droplet Density

The contact angle was measured immediately after preparation of thesurfaces. The results (FIG. 8) show a clear correlation with the molarratio of the mixture of the two SAM components in the solution.

The droplet density is homogeneously distributed over the surface on themacrometer and micrometer scale, so it can be concluded that the two SAMcomponents are homogeneously distributed in this order of magnitude. Thedifference in wettability (contact angle) has only a minor influence ondroplet density (Table 4). An increase in this density is to be expectedif the roughness, surface charge and/or disarrangement of the layersincreases. The low density of the droplets of 100-200 per mm² indicatesthat the layers are relatively well organized and smooth (Tab. 4). Theincorporation of OH-DDPO₄ into the alkyl phosphate layer thus has nomajor influence on the degree of organization of the SAMs.

TABLE 4 Contact angle and droplet density of mixed OH—DDPO₄/DDPO₄ SAM onTa₂O₅; (SD = standard deviation). Vol-% OH—DDPO₄ Contact angle Dropletdensity in solution (±SD) (±SD) 0 110.1 ± 0.8  144 ± 29 10 105.6 ± 1.7 126 ± 26 20 103.6 ± 1.0  104 ± 21 30 86.6 ± 1.4 167 ± 33 40 81.6 ± 1.4125 ± 25 50 73.0 ± 1.8 102 ± 21 60 70.9 ± 1.9  72 ± 30 70 64.1 ± 2.3 161± 32 80 58.2 ± 1.5 123 ± 25 90 57.3 ± 1.2 229 ± 46 100 54.3 ± 3.5 159 ±321.4 Conclusion

It has been shown that alkyl phosphates from aqueous solutions ofcorresponding salts (e.g. ammonium salts) form well-definedself-assembling layers (“SAM”) on a series of metal oxide surfaces.Using XPS, no nitrogen was detected, thus leading to the conclusion thatthese SAMs are especially pure, i.e. that they comprise neither thecation of the salt nor organic solvents. Only a certain proportion ofwater can be expected at the boundary surface. However, this isnoncritical both for biomaterial (implant) and for biosensorapplications.

In the case of pure alkyl phosphate, highly hydrophobic surfaces couldbe produced using the method according to the invention. It was shownhere that alkyl phosphate selectively forms SAMs on transition metaloxides as well as on aluminum oxide, whereas silicon dioxide is notcoated. This opens up the possibility of manufacturing chemicallystructured surfaces on the millimeter to submicrometer or nanometerscale by applying the treatment method of the invention to chemicallystructured surfaces (e.g. using microfabrication techniques such aslithography). For example, any photo or electron beam-lithographicsample with surfaces locally comprising silicon (dioxide) or atransition metal oxide or aluminum oxide may be selectively coated withalkyl phosphates or terminally functionalized alkyl phosphates. Thesilicon dioxide surfaces may in addition be functionalized in a secondstep using a different method. This also includes the possibility ofprecipitating on these surfaces alkyl phosphates or functionalized alkylphosphates from organic solvents, since it is known that silicon dioxidemay also be coated from such nonaqueous solutions. Thus, for example,samples with locally hydrophilic/hydrophobic properties may be produced.It is thus also possible to apply locally differing functional groups.These include different charges (use of e.g. positively charged,terminal amino or ammonium groups and negatively charged carboxy,phosphate or phosphonate groups). A further possibility is the localapplication of protein and cell adhesion-resistant groups (e.g. usingalkyl phosphates terminally modified with oligo-ethylene oxide), whileother zones of the surface have an adhesive character by showingprotein-adsorbing properties. The latter may also be further enhanced byapplying in these zones alkyl phosphates which comprise terminally boundcell-adhesive proteins, such as RGD-containing peptides. In this way itis possible to direct proteins and biological cells selectively tocertain zones of the surface and to use the specific behavior pattern ofsuch separately applied cells. It is likewise possible for biosensorapplications to apply recognition elements, such as antibodies ofproteins, single-strand DNA or RNA chains, etc., locally and selectivelyand thus create the possibility of specifically conducting bioaffinitytests on certain zones of the surface. This is of interest with regardto the “multiarray” technique in sensor technology.

The above descriptions apply by analogy also for salts of alkylphosphonic acids, whose properties, especially with regard to themodification of metal oxide surfaces, are very similar.

EXAMPLE 2 Manufacture of Dodecylphosphate SAM andHydroxydodecylphosphate-SAM as Well as Mixed SAMs on Smooth and RoughMetallic Titanium Implant Surfaces

2.1 Objective and Specification

This example is intended to show that salts of various alkyl phosphoricor alkyl phosphonic acids in aqueous solution are also applicable forthe modification of metallic, oxide-coated implant surfaces using themethod according to the invention. It should further be shown that notonly smooth (e.g. polished), but also rough, topographically structuredsurfaces can be successfully treated.

2.2 Materials and Methods

Substrates: Metal probes of cp (commercially pure) titanium which alwaysshow a naturally formed titanium oxide layer were treated in twodifferent variants as follows:

-   A) Mechanical grinding and polishing, subsequent cleaning in the    organic solvents hexane, acetone and alcohol, followed by    passivation in 30% HNO₃ solution and final cleaning in    oxygen-plasma. This leads to a clean and smooth surface.-   B) Blasting with aluminum oxide particles, followed by chemical    etching in chloride-containing sulfuric acid. This leads to a highly    roughened surface, as shown on scanning electron microscopy (FIG.    9). Such surfaces are preferably used for implants in the skeletal    area, because they show an outstanding capacity for complete    integration in the bone (“osseointegration”).    2.2.1 Alkyl Phosphates

The same materials were used as in Example 1.

2.2.2 Manufacture of Treatment Solutions and Treatments of MetallicSamples.

The same methods were used as in Example 1.

2.3 Results

a) DDPO₄(NH₄)₂ and OH-DDPO₄(NH₄)₂ on Smooth Titanium Surface

Aqueous solutions both of pure DDPO₄(NH₄)₂ and pure OH-DDPO₄(NH₄)₂ andalso different mixture ratios of these two substances were used for thetreatment of smooth and rough titanium surfaces.

Table 5 shows the XPS results of the treated surfaces. As in Example 1,these data show that, with the method according to the invention, alsometallic titanium surfaces (with oxidic passive layer) are occupied bycorresponding molecules in the same way as pure titanium oxide layers.

FIG. 10 shows the corresponding contact angle, measured against water.These are very similar to the correspondingly treated metal oxide-coatedsurfaces (Example 1). Again a characteristic dependence of the contactangle on the composition of the SAM solution is observed. The method isthus also suitable with these materials for the specific adjustment ofsurface-relevant properties (in this case wettability).

TABLE 5 Surface concentration of smooth, metallic titanium surfaces,treated according to the method of the invention with aqueous solutionsof the salts DDPO₄(NH₄)₂, OH—DDPO₄(NH₄)₂ or mixtures thereof indifferent concentration ratios. Vol-% Atomic concentrations OH—DDPO₄(90° exit angle) in solution % C % O (total) % Ti % P 0 46.66 36.5913.76 3 10 55.81 31.06 10.16 2.98 20 57.03 30.49 9.62 2.86 30 48.1136.73 12.24 2.92 40 49.01 36.55 12 2.44 50 54.31 32.49 10.27 2.93 6047.06 37.86 12.61 2.47 70 49.28 36.07 12.1 2.55 80 49.81 35.94 11.822.43 90 49.66 36.58 11.84 1.92 100 45.63 39.32 12.36 2.68b) DDPO₄(NH₄)₂ and OH-DDPO₄(NH₄)₂ on Rough Titanium Surface

Table 6 shows the XPS data of the metallic rough surfaces treated withaqueous solutions of the salts DDPO₄(NH₄)₂ and OH-DDPO₄(NH₄)₂ ormixtures thereof. These samples show a surface structure as illustratedin FIG. 9. Again the surface concentrations indicate a completeformation of the corresponding SAMs. The concentrations here differslightly from those of smooth surfaces, which is attributable to theinfluence of surface topography on the XPS measurement.

FIG. 11 shows the corresponding contact angles as in FIG. 10. Thecontact angles here show a markedly different course compared with thecorresponding curves for smooth surfaces. This is attributable to theinfluence of surface topography. It is known that rough (topographicallystructured), hydrophobic surfaces show markedly higher contact angles(reduced wetting) than smooth hydrophobic surfaces (Ref). This effect isalso observed in nature, and it is from these observations that the nameLotus Effect derives, because, for example, lotus leaves use preciselythis combination of roughness and hydrophobicity to form a self-cleaningsurface.

The high contact angles of the surface treated with DDPO₄(NH₄)₂unequivocally show that these rough surfaces are perfectly coated withthe DDPO₄-SAM.

TABLE 6 Surface concentration of rough, metallic titanium surfaces,treated according to the invention with aqueous solutions of the saltsDDPO₄(NH₄)₂, OH—DDPO₄(NH₄)₂ or mixtures thereof in differentconcentration ratios. Vol-% Atomic concentrations OH—DDPO₄ (90° exitangle) in solution % C % O (total) % Ti % P 0 42.64692 38.87785 15.288333.186893 10 42.01745 39.3542 14.94158 3.686767 20 42.21079 39.239214.57747 3.972532 30 41.14441 40.02102 14.01979 4.814779 40 41.198741.81364 13.49958 3.488083 50 42.50703 40.95192 12.98231 3.55874 6041.44062 42.45984 12.19712 3.902415 70 42.90176 40.70315 12.575573.819517 80 42.77291 40.86229 12.66196 3.702839 90 41.26897 40.9735413.70276 4.054732 100 42.8869 40.12049 13.88565 3.106954

It is thus shown that not only smooth titanium surfaces, but also thosewith a large specific surface and complex roughness, as often used forimplants, e.g. for bone implants, can be successfully coated accordingto the method of the invention.

EXAMPLE 3 Sandwich Immunoassays on Sensor Chips Coated with DDP:Quantitative Determination of the Analytes Immunoglobulin (Rabbit IgG)and the Human Cytokine Interleukin 6 (hIL-6) by Fluorescence Detectionon Planar Waveguides as Sensor Platform

The aim of the experiments was to show that surface modification of aplanar thin-layer waveguide as sensor platform according to theinvention by the application of monolayers of alkyl phosphates orphosphonates can produce a marked increase in sensitivity compared withsensor platforms which have not undergone such pretreatment according tothe invention. The detection sensitivity thus achieved lies in the lowerpg/ml range. Such sensitivity can only be achieved if a correspondinglylarge number of recognition antibodies can be applied to the sensorsurface in active form, i.e. without denaturation. This may be achievedby applying a dodecylphosphate SAM (DDP-SAM). Furthermore, it should bedemonstrated that assays on sensor chips without prior precipitation ofa DDP monolayer, but otherwise with the same pretreatment, showcorrespondingly lower assay signals. Furthermore, it should be shownthat a monolayer of DDP and thus the number of surface-bound recognitionmolecules remains stable over the course of an experiment of severalhours and also that it does not suffer damage as a result of additionalmechanical stress, e.g. as a result of vigorous rinsing of the surfacewith measuring buffer. The stability manifests itself by a constantfluorescence signal response following a binding experiment.

3.1. Sensor Platforms

Rectangular, planar waveguide chips comprising a thin highly refractivewaveguide layer on a 0.7 mm thick transparent substrate with the outerdimensions of 16 mm high×48 mm wide×0.7 mm thick were used as sensorplatforms. The optically transparent substrate material of the sensorchip comprised AF45 glass (refractive index n=1.52 at 633 nm). In thesubstrate were five surface relief gratings with a width of 0.5 mm (inthe direction of propagation of the excitation light to be coupled intothe layer of the sensor platform via the grating structure) andstructured along the full height (48 mm) of the substrate. The gratingswere arranged at intervals of 9 mm along the breadth of the chip. Thearea between two gratings was used as measuring surface. The reliefgratings showed a period of 360 nm and a depth of 12 nm. Thewave-guiding, optically transparent layer comprising Ta₂O₅ had arefractive index of 2.11 at 633 nm (layer thickness 150 nm).

The relief gratings serve to couple laser light of a defined wavelength(here 633 nm) into the sensor layer. Light coupled into the sensor layerand guided there is used for the excitation of fluorophores locatedclose to the surface, i.e. fluorophores located in the evanescent fieldof the guided light. The fluorescence light generated at the surface inthis way serves as a measuring signal for the binding of analytemolecules to specific recognition elements immobilized on the sensorsurface. The number of bound analyte molecules can be determined fromthe intensity of the emitted fluorescence.

A flow cell structure was linked to the pretreated sensor platform forcontact of the analysis sample with the sensor chip. The flow cellstructure comprised sample vessels in each of which there was ameasuring surface between two gratings on the chip. Each sample vesselhad a volume of 15 μl, with a base area of 7 mm×11 mm on the sensorsurface and a cell height of 0.2 mm. The analysis sample could be addedto or removed from the cell via an injection or drainage channel(diameter 0.5 mm). The sample/buffer was added by means of a dispensingpump via hose connections fitted between the pump and the inlet.

3.2. Chip Pretreatment

Before being joined together with the flow cell structure, the sensorsurface was cleaned by means of a wet chemical process first withisopropanol several times, then with concentrated sulfuric acidcontaining 2.5% ammonium peroxodisulfate. A monomolecular layer(monolayer) of dodecylphosphate (DDP) was then precipitated on thehydrophilic waveguide surface from aqueous phase in a self-assemblyprocess. A 0.5 mM DDP/ammonium salt solution in water was used for theprecipitation. The sensor surface was incubated for 2 h in the DDPsolution under constant agitation at room temperature, then thoroughlywashed in running water and dried under a stream of nitrogen. Theprecipitation of the DDP monolayer led to a hydrophobic surface (contactangle about 110° with water).

3.3. Application of Biological Recognition Elements

(A) For Detection of Rabbit IgG

The sensor surface was incubated with an aqueous solution ofbiotinylated bovine serum albumin (BSA-biotin, Sigma, catalog no.A-6043). A 100 μg/ml solution in phosphate-buffered saline solution(PBS), pH 7.4, was used. The chip was incubated for 1 h at roomtemperature, thoroughly washed under running, deionized water(Millipore, spec. resistance=18 mΩ cm) and then dried under a stream ofnitrogen. The surface was then blocked using a solution of 1 mg/ml purebovine serum albumin (BSA, Sigma, catalog no. A7906) in PBS, pH 7.4.Incubation was resumed for 15 min, and the chip was again washedthoroughly under running, deionized Millipore water and then dried undera stream of nitrogen. The blocked chip was then incubated in the samemanner with 50 μg/ml streptavidin (Sigma catalog no. S4762) in PBS, pH7.4, for 30 min. The streptavidin molecules here bind to the availablebiotin groups of the albumin. Surplus streptavidin was removed again byrising with water. Finally, the surface was incubated with thebiotinylated recognition antibody anti-rabbit IgG (Sigma, catalog no.B-7389) in a concentration of 12 μg/ml in PBS, pH 7.4, for 30 min,thoroughly washed again and dried with nitrogen. The dry chip was thenconnected to the flow cell structure.

(B) For Detection of hIL-6

The sensor surface was incubated in the region of the measuring surfacewith 50 μl of an aqueous solution of monoclonal mouse anti-hIL-6antibody (R&D Systems, catalog no. mAb206). The freeze-dried mouseanti-hIL-6 antibody was reconstituted in a concentration of 0.5 mg/ml inten percent, phosphate-buffered saline solution (10% PBS), pH 7.4, andthen diluted with 10% PBS, pH 7.4, to a concentration of 10 μg/ml. Withthis solution, the chip was incubated for 2 h at room temperature,thoroughly washed under running, deionized Millipore water (spec.resistance=18 mΩ cm) and then dried under a stream of nitrogen. Thesurface was then blocked using a solution of 1 mg/ml bovine serumalbumin (BSA, Sigma, catalog no. A7906) in PBS, pH 7.4. Incubation wasresumed for 15 min, and the chip was again washed thoroughly underrunning, deionized Millipore water and then dried under a stream ofnitrogen. The dried chip was connected to the flow cell structure andinserted into the measuring apparatus.

3.4. Analytical Measuring Apparatus

The sensor chip is mounted on a computer-controlled adjustment unitwhich permits translation parallel with and perpendicular to the gratinglines as well as rotation about an axis parallel with the grating linesof the sensor platform. Immediately behind the laser used as excitationlight source is a shutter in the light path designed to block this lightpath when no measurement data are to be recorded. In addition, neutralfilters or polarizers may be placed here or also at other positionsalong the path of the excitation light to the sensor platform to achievestepped or continuous variations in the intensity of the excitation.

The excitation light beam of a helium neon laser at 632.8 nm(Melles-Griot 05-LHP-901, 1.1 mW) is expanded in one dimension with alens system using a cylinder lens and directed through a slit aperturemeasuring 0.5 mm×7 mm to produce a parallel light bundle almostrectangular in cross-section and almost homogeneous in cross-sectionintensity. Thereby, the polarization of the laser is orientated inparallel with the grating lines of the sensor platform for excitation ofthe TE₀ mode under coupling conditions. The excitation light is directedthrough the rear aspect of the sensor platform, i.e. through theoptically transparent layer (b) onto a in-coupling grating within one ofthe 5 sample vessels, wherein the in-coupling grating in a sample vesselunder the conditions in the example is located on the left margin of therectangular sample cell. The angle between the sensor platform and theincoming excitation light bundle is adjusted for maximum coupling intothe optically transparent layer (a) by rotation about the aforementionedaxis. With the aforementioned parameters of the sensor platform, theresonance angle for the in-coupling in air was about 2.6°.

A red-sensitive photomultiplier tube (PMT) (H6240, Hamamatsu, Japan),which is used in single-photon counting mode with a 225 MHz counter(model 53131A, Hewlett-Packard, USA), serves as detector. The signalrecording and focusing of the fluorescence light emitted from thesubstrate side of the sensor chip onto the detection window of the PMTtake place using an objective of own construction (imaging ratio 1:1,numerical aperture 0.19). In the parallel section of the light path ofthe objective, mounted on a filter changer, are two interference filters(Omega, Brattleborough, Vermont) with a central wavelength of 680 nm and40 nm bandwidth, as well as a thin glass plate in front of the filterunit as beam splitter, which can detect excitation light emitted fromthe sensor surface as a reference signal via measurement with aphotodiode. This arrangement ensures that the luminescence signal andthe reference signal originate from the same measuring range. Thereference signals (at the excitation wavelength) and the actualmeasuring signal (at the luminescence wavelength) can be measuredsimultaneously. The measuring signals displayed have already beencorrected by means of the reference signal in order to eliminatevariations in the measuring range. The measuring signals were determinedas a quotient of the luminescence signal and the reference signal,multiplied by the factor 10₆.

3.5. Performance of the Analytical Detection Method

The sandwich immunoassay format was selected for the specificrecognition of the analytes to be detected (rabbit IgG and h-IL-6).

3.5.1. Sample Preparation

Of the analytes to be quantified (rabbit IgG and h-IL-6) standardsolutions of 500 μl in PBS, pH 7.4, with 0.1% BSA and 0.05% Tween20 wereprepared in each case. The concentrations in each case amounted to 0,0.067, 0.67, 6.7, 67, and 670 pM for the analyte rabbit IgG (Sigma,catalog no. 1-5006) and 0, 0.5, 1, 2.5, 5, 25, 50, 250, 500, 2500, 10000pg/ml for the analyte hIL-6 (R&D Systems, catalog no. 206-IL-06) withthe addition in each case of 10% calf serum (Anawa, catalog no.8270-0209). These standard solutions were intended for generatingcalibration curves of the analytes in order to determine theconcentrations of samples of unknown analyte quantity.

The calibration solutions and also the samples with unknown analyteconcentrations to be determined were then mixed in each case with 500 μlof a solution comprising polyclonal tracer antibodies. Anti-rabbit IgGfrom goat, labeled with Cy5-dye (Amersham-Pharmacia, catalog no.PA45004), with a dye-to-protein ratio of 4:1, was used for the detectionof rabbit IgG. Anti-hIL-6 antibody (AF-206-NA, R&D Systems, UK) labeledwith Cy5 and having a dye-to-protein ratio of 5:1 was used for thedetection of hIL-6. Analyte solution and tracer solution were each mixedin the ratio 1:1 and incubated for 1 h in the dark at 37° C.

3.5.2. Performance of Measurement

The pretreated, dry sensor chip with applied flow cell structure wasplaced in the measuring device. To perform an experiment, the dry samplevessel was rinsed with a continuous flow of measuring buffer (PBS, pH7.4, 0.05% Tween 20, 0.1% BSA) at 0.25 ml/min for 5 min. To optimize theexcitation conditions for luminescence excitation, the sensor chip withthe sample cells located thereon and the solution located therein wasadjusted for maximum in-coupling of the excitation light via the gratingstructure assigned to the respective measuring region, and the intensityof the light emitted from the measuring region was measured over aperiod of 1s. The preincubated standard solutions were added to thesample cell sequentially in ascending concentration from an autosampler(231XL, Gilson, USA) by means of a dispensing pump. On rinsing, residualsolution was eliminated via the drainage system of the sample cell.

In the case of the detection of rabbit IgG, the total sample (1 ml) wasapplied under continuous flow (0.25 ml/min) for 4 min and during thisperiod the binding of the analyte to the prepared sensor surface wasmeasured in real time by means of the increase in fluorescence(measurement points every 30 s, FIG. 12). After then rinsing in eachcase for 8 min with buffer solution, the sample was applied in the nexthigher concentration. In the case of the detection of hIL-6, the samplewas injected only briefly and rapidly (flow rate 1 ml/min), and thebinding of the analyte molecules from the standing sample to theimmobilized recognition elements thereof was then measured without flowfor 30 min until a steady state was reached (measurement points every 60s, FIG. 13 a). In this case there was no rinsing step between sequentialsample additions. The corrected signal curve, from the quotient of themeasured fluorescence and reference signals, is shown in FIGS. 12(rabbit IgG as analyte) and 13a (hIL-6 as analyte). The extent ofnonspecific binding was determined in each case from the firstmeasurement of a sample without analytes. The net binding signals forthe different analyte concentrations were then determined as thedifference between the corrected gross signal measured at the currentanalyte concentration and the nonspecific binding signal. From this, thesignal concentration curves for rabbit IgG (FIG. 13 b for hIL-6) weregenerated.

FIG. 13 a shows the curve of the binding signals in a measurement seriesfor detection of hIL-6. Throughout the experiment, which lasted morethan 7 hours, no systematic decrease in signal height was observed, evenafter the saturation signals had been reached in the stationary statefor the analyte concentration concerned. This means that no signal lossoccurred either due to fading of the dye or to possible detachment ofsignal-emitting binding complexes from the surface, which indicates avery stable immobilization of the biological recognition elements usedon the surface of the sensor platform. Vigorous rinsing (flow rate 2ml/min for 5 min) of the sensor surface with relatively large quantitiesof sample, equivalent to the application of mechanical stress, also ledto a stationary signal and not to a decrease in the signal, which is afurther indication of the high degree of stability of theimmobilization.

Performance hIL-6 Assay without Coupling Layer

To study the positive influence of a DDP monolayer on the intensity ofthe assay signals, i.e. the quantity of functionally immobilizedrecognition elements, and thus on the attainable limits of detection,the hIL-6 immunoassay on sensor chips was carried out for comparisonpurposes without an applied DDP monolayer, but with the surfaceotherwise pretreated in the same way. As shown in FIG. 13 a, the bindingsignals did not attain more than 10% of the signals from experimentsusing precipitated adhesion-promoting layer of DDP. The lower signalsare attributable to a lower surface occupancy and/or to denaturation ofthe indicator antibodies on the untreated surface.

EXAMPLE 4 Sandwich Immunoassays on Monolayers of MixedHydrophobically/Hydrophilically Terminated Alkyl Phosphates

The aim of the experiment was to show that the assay signals and thusdetection sensitivities of immunoassays can be substantially increasedon sensor surfaces which are produced by precipitation of monolayers ofdefined mixtures of hydrophobically (alkyl) and hydrophilically(hydroxyl) terminated alkyl phosphates. As already shown, the contactangle of metal oxide surfaces with water can be selectively adjusted byappropriate mixing of hydrophilically and hydrophobically terminatedalkyl phosphates. This permits a specific optimization of the number offunctionally immobilized recognition elements, depending on the natureof the recognition element used. The antibodies described in Example 3were used as recognition elements.

Sensor chip surfaces were prepared with monolayers of aqueous mixturesof DDP-OH:DDP in the ratio of 0:100, 20:80, 40:60, 60:40, 80:20 and100:0 percent. The chips were further treated and the measurementsconducted as described in Example 3. FIG. 14 shows the standardizedsignal responses for the mixtures used. In the case of the detection ofrabbit IgG, the signal response for the optimum mixture may be increasedby up to 300% over the value with a pure DDP layer. The optimum mixtureratio lies between 80:20 and 60:40 percent DDP-OH:DDP, thus with more ofa hydrophilic surface. Similar values have been achieved in the assaysfor the detection of hIL-6.

EXAMPLE 5 Production of a Biocompatible Double Layer asAdhesion-Promoting Layer (comprising a DDP and a Lipid Layer) Free fromOrganic Solvent Residues

As already shown, very hydrophobic monolayers on metal oxide surfacescan be produced by precipitation of DDP from aqueous phase. Suchhydrophobic, self-assembled layers are highly suited to theprecipitation of further monolayers, e.g. comprising natural orsynthetic lipids. Double layers thus generated simulate on their surfacethe properties of natural cell membranes and are especially suitable forthe functional immobilization of membrane-anchored or membrane-boundrecognition elements such as cell receptors or even whole membraneparticles, which are known to be very sensitive and to respond tocontact with media having physicochemical properties other than those ofthe natural membrane environment by undergoing changes. Thus thesmallest changes in protein structure resulting from contact with ahydrophobic, hard sensor surface can lead to a total failure of receptorfunction.

Part A) Production of Biocompatible Double Layers Comprising a PrimaryDDP Monolayer with a Hydrophobic Surface and a Secondary Lipid Monolayerwhich has Been Produced by Spontaneous Spreading of Lipid Vesicles onthe Primary Hydrophobic Surface.

According to the description in Example 3, hydrophobic sensor surfaceswere produced with a DPP monolayer. Lipid vesicles were produced fromsynthetic lipid 1-palmitoyl(C₁₆)-2-oleoyl(C₁₈)—SN-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids, catalogno. 850457). To this end, 6 mg of dry POPC was taken up in 3 ml ofvesicle buffer (150 mM NaCl, 10 mM phosphate buffer, 0.002% NaN₃, pH7.5), incubated for about 12 h in the refrigerator at 4° C. and thenmixed, which resulted in a turbid suspension. Monolayered lipid vesiclesmeasuring about 100 nm were then produced by extrusion (about 20 times)through a 100 nm pore filter (extruder ?). Vesicle formation wasobserved on the basis of an increasing clarification of the suspension.The hydrophobic surfaces of the sensor chips were incubated with dilutedvesicle suspension (0.5 mg/ml) for one hour at room temperature withsample vessels fitted. Surplus vesicle suspension is eliminated bythorough rinsing with vesicle buffer. The kinetics underlying theformation of a second monolayer of lipids by vesicle spreading wasobserved using fluorescently labeled vesicles comprising 0.1% of a lipidlabeled with dye (1,2-dioleoyl(C₁₈)—SN-glycero-3-phosphoethanolaminefluorescein (DOPE fluorescein) by means of fluorescence measurement inthe measurement apparatus described in Example 3.4. The kineticsunderlying the formation of the second lipid layer (with 0.1% lipid dye)on the DDP layer is shown in FIG. 15. The signal change between initialbaseline and final signal level after rinsing of the surface correspondsto the signal of a monolayer. This signal amounts to about 50% of thesignal upon the formation of a double layer of lipids fluorescentlylabeled in the same way when a similar vesicle suspension is incubatedwith a blank, hydrophilic metal oxide layer as sensor surface (withouthydrophobic primary layer). Part B) Demonstration of theBiocompatibility of a DDP-Lipid Double Layer for the Immobilization ofMembrane Receptors Detected by Means of a Binding Assay Using NativeMembrane Fragments Immobilized on the Double Layer with Coupled MembraneEnzyme Na,K-Atpase.

In its function as an ion pump (of K⁺ and Na⁺ ions), the enzymeNa,K-ATPase—an integral component of physiological cellmembranes—regulates the membrane potential of living cells. Afluorescence-based assay was developed for the specific binding of K⁺ions to the enzyme on sensor surfaces. The K⁺ binding signal serves as asensitive measure of enzyme function, i.e. the pumping of K⁺ ions acrossthe cell membrane. The ions required for the pumping process areprovided by the specific binding of these ions to the protein.

Physiological membrane fragments which had coupled the membrane enzymeNa,K-ATPase to their membrane were immobilized on a double-layer systemdescribed in Part A, a DDP (from aqueous phase) and lipid layer on aplanar waveguide chip as sensor platform. The functional and stableimmobilization of membrane fragments and thus of the enzymes isdemonstrated by the ability to generate K⁺ binding signals describedabove. Upon otherwise comparable immobilization of membrane fragments onblank sensor chips without a double-layer system, the ability togenerate the K⁺ binding signal is lost. The biocompatible effect of theadhesion-promoting layer presented—in this case a double-layer systemwhich is free of organic solvent residues and comprises a layer of alkylphosphates and lipids respectively—on the functional immobilization ofmembrane proteins and whole membrane fragments is thus demonstrated. Theimmobilization of the membrane enzyme with the possibility of contactwith the blank sensor surface leads to denaturation and thus loss offunction of the protein to be studied.

BRIEF DESCRIPTION OF THE DRAWINGS:

FIG. 1: Schematic representation of the ordered structure of alkylphosphate SAMs on an oxide surface

FIG. 2: Binding conditions in the assembled SAM layer between alkylphosphates and a tantalum dioxide (Ta₂O₅) surface

FIG. 3: Measurements of droplet density on DDPO₄ SAMs on differentsmooth metal oxide surfaces (image size: 1 mm²): a) Al₂O₃, b) Ta₂O₅, c)Nb₂O₅, d) ZrO₂, e) Fe₂O₃, f) TiO₂, g) TiO_(0.4)Si_(0.6)O₂ and h) SiO₂.See text.

FIG. 4: DDPO₄ SAM on the Ti_(0.4)Si_(0.6)O₂ surface with a 2 mm widestrip of Fe₂O₃. The Fe₂O₃ surface is covered with DDPO₄ and ishydrophobic (low droplet density), whereas the substrate(Ti_(0.4)Si_(0.6)O₂) remains more hydrophilic, since it is notcompletely covered with DDPO₄.

FIG. 5 a: O1s XPS signal of OH-DDPO₄ SAMs on Ta₂O₅ substrates: The O1sspectra at the different exit angles (20.5°=top curve a; 11.5°=middlecurve b) are compared with the corresponding signal of the DDPO₄ SAM(bottom curve c).

FIG. 5 b: O1s XPS signal of OH-DDPO₄ SAMs on Nb₂O₅ substrates: The O1sspectra at the different exit angles (20.5°=top curve a; 11.5°=middlecurve b) are compared with the corresponding signal of the DDPO₄ SAM(bottom curve c).

FIG. 6 a: Comparison of the carbon concentration, determined by XPS, ofOH-DDPO₄ and DDPO₄ SAMs on Ta₂O₅, as a function of the sine of detectionangle Θ. (OH-DDPO₄ carbon: solid circles; DDPO₄ carbon: open squares.)

FIG. 6 b: Comparison of the carbon concentration, determined by XPS, ofOH-DDPO₄ and DDPO₄ SAMs on Nb₂O₅, as a function of the sine of detectionangle Θ). (OH-DDPO₄ carbon: solid circles; DDPO₄ carbon: open squares.)

FIG. 7 a: XPS peak areas as a function of the sine of detection angle Θof Nb(3d), C(1s) and P(2p) on Nb₂O₅. The O(1s) signal was deconvolutedinto three components: O(Nb₂O₅): squares, O(PO₄): triangles, O(ROH):circles. Similar results were also found in the case of the substrateTa₂O₅.

FIG. 7 b: XPS peak areas as a function of the sine of detection angle Θof the various oxygens for OH-DDPO₄ SAM on Nb₂O₅. The O(1s) signal wasdeconvoluted into three components: O(Nb₂O₅): squares, O(PO₄):triangles, O(ROH): circles. Similar results were also found in the caseof the substrate Ta₂O₅.

FIG. 8. Contact angle of mixed OH-DDPO₄/DDPO₄ SAMs on Ta₂O₅. The resultsare presented as a function of the mixture ratio in the SAM solution(vol-%). The total alkyl phosphate concentration was kept constant (0.5mM).

FIG. 9 Surface of a pure titanium (cp Ti) sample roughened by blastingand chemical etching. Origin of sample: Institut Straumann AG,Waldenburg, Switzerland. Scanning electron microscopy.

FIG. 10. Contact angle as a measure of the wettability of smooth,metallic titanium surfaces, treated with aqueous solutions of the saltsDDPO₄(NH₄)₂, OH-DDPO₄(NH₄)₂ and mixtures thereof (upper data series:advancing contact angle, lower data series: receding contact angle).

FIG. 11. Contact angle as a measure of the wettability of rough,metallic titanium surfaces, treated with aqueous solutions of the saltsDDPO₄(NH₄)₂, OH-DDPO₄(NH₄)₂ and mixtures thereof (upper data series:advancing contact angle, lower data series: receding contact angle).

FIG. 12: Corrected signal curve upon the addition of increasingconcentrations of the analyte rabbit IgG, detected by means offluorescence-based sandwich assay on thin-layer waveguides as sensorplatforms, on which the specific recognition antibodies have beenimmobilized on a monolayer of dodecylphosphate (DDP). In the sequentialassay steps, the addition of the analyte over a period of 4 min isfollowed in each case by rinsing with buffer solution (8 min).

FIG. 13 a: Corrected signal curve upon the addition of increasingconcentrations of the analyte hIL-6, detected by means offluorescence-based sandwich assay on thin-layer waveguides as sensorplatforms, on which the specific recognition antibodies have beenimmobilized in the presence and the absence of a monolayer ofdodecylphosphate (DDP). Clearly visible here are the marked signalincreases in the presence of a DDP monolayer, indicating a functionalimmobilization of a large number of recognition antibodies.

FIG. 13 b: Signal-concentration function (dose-response curve) generatedfrom the stationary signal responses according to FIG. 13 a (chip withDDP monolayer). The course of the data points corresponds to a Langmuirbinding isotherm (1:1 binding), which was fitted to the data.

FIG. 14: Relative assay signals of experiments for the detection ofrabbit IgG (250 μM), carried out on chip surfaces with monolayersprecipitated from different, aqueous mixtures of OH-terminated DDP andhydrophobically terminated DDP. The error bars show the deviations ofmeasurements on two chips. Values were standardized to the signals at60% OH-terminated DDP portion.

FIG. 15: Signal curve during the formation of a second monolayer offluorescently labeled lipids (POPC with 0.1% DOPE fluorescein) byspreading of lipid vesicles (diameter about 110 nm) on a hydrophobic DDPmonolayer on a metal oxide thin-layer waveguide as sensor platform.

1. A method which comprises precipitating mono or multiple layers oforganophosphoric acids of the general formula I (A)Y—B—OPO₃H₂  (IA) or of organophosphonic acids of the general formula I(B)Y—B—PO₃H₂  (IB) and the salts thereof, on a substrate surface of pure ormixed oxides, nitrides or carbides of metals or semiconductors, whereinB is an alkyl, alkenyl, alkinyl, aryl, aralkyl, hetaryl or hetarylalkylresidue and Y is hydrogen or a functional group from the hydroxy,carboxy, amino, optionally low-alkyl-substituted mono or dialkylaminoseries, thiol, or a negative acid group from the ester, phosphate,phosphonate, sulfate, sulfonate, maleimide, succinimydyl, epoxy,acrylate series, wherein a biological, biochemical or syntheticrecognition element is coupled to B or Y by addition or substitutionreaction, wherein compounds may also be added conferring on thesubstrate surface a resistance to protein adsorption and/or to celladhesion and the B chain may optionally be comprised of one or moreethylene oxide groups, rather than one or more —CH2— groups, and whereinthe organophosphoric acid or organophosphonic acid is precipitated fromwater-soluble salts of a compound of formula (IA) or (IB) for thetreatment of these surfaces, of sensor platforms, implants and medicalaccessory devices, and wherein the biochemical, biological or syntheticrecognition elements (coupled to B or Y) are selected from among thegroup of nucleic acids, such as DNA, RNA, oligonucleotides, nucleic acidanalogs, such as PNA, monoclonal or polyclonal antibodies, peptides,enzymes, aptamers, synthetic peptide structures, soluble membrane-boundproteins and proteins isolated from a membrane, such as receptors,ligands thereof, antigens for antibodies, biotin, “histidine tagcomponents” and complexing partners thereof.
 2. A method which comprisesprecipitating mono or multiple layers of organophosphoric acids of thegeneral formula I (A)Y—B—OPO₃H₂  (IA) or of organophosphonic acids of the general formula I(B)Y—B—PO₃H₂  (IB) and the salts thereof, on a substrate surface of pure ormixed oxides, nitrides or carbides of metals or semiconductors, whereinB is an alkyl, alkenyl, alkinyl, aryl, aralkyl, hetaryl or hetarylalkylresidue and Y is hydrogen or a functional group from the hydroxy,carboxy, amino, optionally low-alkyl-substituted mono or dialkylaminoseries, thiol, or a negative acid group from the ester, phosphate,phosphonate, sulfate, sulfonate, maleimide, succinimydyl, epoxy,acrylate series, wherein a biologically effective recognition element iscoupled to B or Y by addition or substitution reaction, whereincompounds may also be added conferring on the substrate surface aresistance to protein adsorption and/or to cell adhesion and the B chainmay optionally be comprised of one or more ethylene oxide groups, ratherthan one or more —CH2— groups, and wherein the organophosphoric acid ororganophosphonic acid is precipitated from water-soluble salts of acompound of formula (IA) or (IB) for the treatment of these surfaces, ofsensor platforms, implants and medical accessory devices, and whereinthe biologically effective recognition element comprises peptides,proteins, glycoproteins, growth factor, such as TGF-β, or BMP (bonemorphogenic protein).
 3. A method which comprises precipitating mono ormultiple layers of organophosphoric acids of the general formula I (A)Y—B—OPO₃H₂  (IA) or of organophosphonic acids of the general formula I(B)Y—B—PO₃H₂  (IB) and the salts thereof, on a substrate surface of pure ormixed oxides, nitrides or carbides of metals or semiconductors, whereinB is an alkyl, alkenyl, alkinyl, aryl, aralkyl, hetaryl or hetarylalkylresidue and Y is hydrogen or a functional group from the hydroxy,carboxy, amino, optionally low-alkyl-substituted mono or dialkylaminoseries, thiol, or a negative acid group from the ester, phosphate,phosphonate, sulfate, sulfonate, maleimide, succinimydyl, epoxy,acrylate series, wherein a biological, biochemical or syntheticindicator element may be coupled to B or Y by addition or substitutionreaction, wherein compounds are also added conferring on the substratesurface a resistance to protein adsorption and/or to cell adhesion andthe B chain may optionally be comprised of one or more ethylene oxidegroups, rather than one or more —CH2— groups, and wherein theorganophosphoric acid or organophosphonic acid is precipitated fromwater-soluble salts of a compound of formula (IA) or (IB) for thetreatment of these surfaces, of sensor platforms, implants and medicalaccessory devices, and wherein the compounds which confer on thesubstrate surface a resistance to protein adsorption and/or to celladhesion are selected from the group of compounds which are formed fromoligo(ethylene oxide), phosphoryl choline, heparin, saccharides,albumins, especially bovine serum albumin or human serum albumin,casein, nonspecific, polyclonal or monoclonal, heterologous or for theanalyte or analytes to be determined empirically nonspecific antibodies(especially for immunoassays), detergents (such as Tween 20), fragmentednatural DNA or synthetic DNA not hybridizing with polynucleotides foranalysis, such as a herring or salmon sperm extract (especially forpolynucleotide hybridization assays), or also uncharged, but hydrophilicpolymers, such as polyethylene glycols or dextrans.
 4. A method whichcomprises precipitating mono or multiple layers of organophosphoricacids of the general formula I (A)Y—B—OPO₃H₂  (IA) or of organophosphonic acids of the general formula I(B)Y—B—PO₃H₂  (IB) and the salts thereof, on a substrate surface of pure ormixed oxides, nitrides or carbides of metals or semiconductors, whereinB is an alkyl, alkenyl, alkinyl, aryl, aralkyl, hetaryl or hetarylalkylresidue and Y is hydrogen or a functional group from the hydroxy,carboxy, amino, optionally low-alkyl-substituted mono or dialkylaminoseries, thiol, or a negative acid group from the ester, phosphate,phosphonate, sulfate, sulfonate, maleimide, succinimydyl, epoxy,acrylate series, wherein a biological, biochemical or syntheticrecognition element is coupled to B or Y by addition or substitutionreaction, wherein compounds are also added conferring on the substratesurface a resistance to protein adsorption and/or to cell adhesion in aSAM molecule and the B chain may optionally be comprised of one or moreethylene oxide groups, rather than one or more —CH2— groups, and whereinthe organophosphoric acid or organophosphonic acid is precipitated fromwater-soluble salts of a compound of formula (IA) or (IB) for thetreatment of these surfaces, of sensor platforms, implants and medicalaccessory devices.
 5. A sensor platform having a surface of a mono ormultiple layers of organophosphates and/or organophosphonates.
 6. Asensor platform according to claim 5 comprising at least one array ofbiological or biochemical or synthetic recognition elements, immobilizedin discrete measurement areas (d) for the specific recognition and/orbinding of one or more analytes and/or specific interaction with saidanalytes.
 7. A sensor platform according to claim 5 capable of detectionof one or more analytes by means of labels selected from the groupconsisting of luminescence labels, especially luminescent intercalatorsor “molecular beacons”, absorption labels, mass labels, especially metalcolloids or plastic beads, spin labels, such as ESR or NMR labels, andradioactive labels.
 8. A sensor platform according to claim 6 capable ofdetection of analyte based on the determination of a change in theeffective refractive index as a result of molecular adsorption ordesorption on the measurement areas (d).
 9. A sensor platform accordingto claim 8 capable of detection of analyte based on determination of achange in conditions for generating a surface plasmon in a metal layerof a multiple layer system, wherein the metal layer preferably comprisesgold or silver.
 10. A sensor platform according to claim 6 capable ofdetection of analyte based on the determination of a change in one ormore luminescences.
 11. A sensor platform according to claim 10 capableof delivery of excitation light in a vertical illuminator.
 12. A sensorplatform according to claim 6, wherein material of the sensor platformthat is in contact with the measurement areas is transparent orabsorbent within a depth of at least 200 nm from the measurement areasin at least one excitation wavelength.
 13. A sensor platform accordingto claim 11, wherein the delivery of the excitation light is in atransmission configuration.
 14. A sensor platform according to claim 13,wherein material of the sensor platform is transparent in at least oneexcitation wavelength.
 15. A sensor platform according to claim 5 formedas an optical waveguide which is preferably essentially planar.
 16. Asensor platform according to claim 15 which comprises an opticallytransparent material selected from the group of silicates, e.g. glass orquartz, transparent thermoplastic or moldable plastic, for examplepolycarbonate, polyimide, acrylates, especially polymethylmethacrylate,and polystyrene.
 17. A sensor platform according to claim 15 whichcomprises an optical thin-layer waveguide with a layer which istransparent in at least one excitation wavelength (a) on a layer whichis likewise transparent in at least this excitation wavelength (b) witha lower refractive index than layer (a).
 18. A method according to claim1, wherein the mono or multiple layers of compounds of formula (IA)and/or (IB) are free of organic solvents.
 19. A method according toclaim 1, wherein the water-soluble salts of a compound of formula (IA)or (IB) are sodium, potassium and/or ammonium salts.
 20. A methodaccording to claim 1, wherein the pure or mixed oxides, nitrides orcarbides of metals are solid bodies or layers on substrates of any kindand the metals are selected from the group consisting of tantalum,niobium, titanium, vanadium, zirconium, hafnium, molybdenum, tungsten,silicon and mixtures thereof.
 21. A method according to claim 2, whereinthe mono or multiple layers of compounds of formula (IA) and/or (IB) arefree of organic solvents.
 22. A method according to claim 2, wherein thewater-soluble salts of a compound of formula (IA) or (IB) are sodium,potassium and/or ammonium salts.
 23. A method according to claim 2,wherein the pure or mixed oxides, nitrides or carbides of metals aresolid bodies or layers on substrates of any kind and the metals areselected from the group consisting of tantalum, niobium, titanium,vanadium, zirconium, hafnium, molybdenum, tungsten, silicon and mixturesthereof.
 24. A method according to claim 3, wherein the mono or multiplelayers of compounds of formula (IA) and/or (IB) are free of organicsolvents.
 25. A method according to claim 3, wherein the water-solublesalts of a compound of formula (IA) or (IB) are sodium, potassium and/orammonium salts.
 26. A method according to claim 3, wherein the pure ormixed oxides, nitrides or carbides of metals are solid bodies or layerson substrates of any kind and the metals are selected from the groupconsisting of tantalum, niobium, titanium, vanadium, zirconium, hafnium,molybdenum, tungsten, silicon and mixtures thereof.
 27. A methodaccording to claim 4, wherein the mono or multiple layers of compoundsof formula (IA) and/or (IB) are free of organic solvents.
 28. A methodaccording to claim 4, wherein the water-soluble salts of a compound offormula (IA) or (IB) are sodium, potassium and/or ammonium salts.
 29. Amethod according to claim 4, wherein the pure or mixed oxides, nitridesor carbides of metals are solid bodies or layers on substrates of anykind and the metals are selected from the group consisting of tantalum,niobium, titanium, vanadium, zirconium, hafnium, molybdenum, tungsten,silicon and mixtures thereof.